Herbicide-resistant rice plants, polynucleotides encoding herbicide-resistant acetohydroxyacid synthase large subunit proteins, and methods of use

ABSTRACT

Herbicide-resistant rice plants, isolated polynucleotides that encode herbicide resistant and wild-type acetohydroxy-acid synthase large subunit 1 (AHASL1) polypeptides, and the amino acid sequences of these polypeptides, are described. Expression cassettes and transformation vectors comprising the polynucleotides of the invention, as well as plants and host cells transformed with the polynucleotides, are described. Methods of using the polynucleotides to enhance the resistance of plants to imidazolinone herbicides, and methods for controlling weeds in the vicinity of herbicide-resistant plants are also described.

FIELD OF THE INVENTION

This invention relates to the field of agricultural biotechnology,particularly to herbicide-resistant rice plants and to novelpolynucleotide sequences that encode herbicide-resistant acetohydroxyacid synthase large subunit proteins.

BACKGROUND OF THE INVENTION

Weeds are one of the major constraints to rice production. Directseeding has reduced the labor problems of transplanting; however, thistechnology has helped to increase the weed problem. Herbicide use inrice is a common practice in most of the rice regions under directseeding crop and/or developed countries that grow rice under eithertransplanting or direct seeding systems. Usually a grass and a broadleafherbicide are applied one or more times in order to control weeds inrice crops.

Grasses, sedges and weedy rice (red rice) have been the major groups ofspecies that possess high fitness to the same environments where rice isgrown. They have become globally distributed and are difficult tocontrol weeds in rice crops. Although there are several culturalpractices that aid in control of weeds and are convenient for betterenvironmental care, these practices impose restrictions and increaseproduction costs. Land preparation, land leveling, levees and depth ofwater, land rotation, certified seed, proper plant systems and dates ofplanting could be some of the cultural practices that may help to reducethe weed seed bank and the development of herbicide-tolerant weeds.

In spite of the many recommendations for better cultural practices thefarmers still rely on the use of herbicides as the main tool to controlweeds. The use and abuse of some of these chemicals has resulted in thedevelopment of tolerant weeds like propanil-resistant andbutachlor-resistant barnyardgrass (Echinochloa crus galli). In thesecases, it would be convenient to have other herbicides with differentmodes of action with the ability to control most of these weed species.The availability of such herbicides would allow for a rotation ofherbicides with a different mode of action than those herbicides thatare commonly used in rice production.

Imidazolinones are a group of herbicides with a different mode of actionthan the commonly used rice herbicides. These herbicides are known toinhibit acetohydroxyacid synthase (AHAS; EC 4.1.3.18, also known asacetolactate synthase or ALS), a key enzyme for the biosynthesis ofbranched-chain amino acids. Inn particular, acetohydroxyacid synthase(AHAS; EC 4.1.3.18, also known as acetolactate synthase or ALS), is thefirst enzyme that catalyzes the biochemical synthesis of the branchedchain amino acids valine, leucine and isoleucine (Singh (1999)“Biosynthesis of valine, leucine and isoleucine,” in Plant Amino Acid,Singh, B. K., ed., Marcel Dekker Inc. New York, N.Y., pp. 227-247). AHASis the site of action of four structurally diverse herbicide familiesincluding the sulfonylureas (LaRossa and Falco (1984) Trends Biotechnol.2:158-161), the imidazolinones (Shaner et al. (1984) Plant Physiol.76:545-546), the triazolopyrimidines (Subramanian and Gerwick (1989)“Inhibition of acetolactate synthase by triazolopyrimidines,” inBiocatalysis in Agricultural Biotechnology, Whitaker, J. R. and Sonnet,P. E. eds., ACS Symposium Series, American Chemical Society, Washington,D.C., pp. 277-288), and the pyrimidyloxybenzoates (Subramanian et al.(1990) Plant Physiol. 94: 239-244.). Imidazolinone and sulfonylureaherbicides are widely used in modern agriculture due to theireffectiveness at very low application rates and relative non-toxicity inanimals. By inhibiting AHAS activity, these families of herbicidesprevent further growth and development of susceptible plants includingmany weed species. Several examples of commercially availableimidazolinone herbicides are PURSUIT® (imazethapyr), SCEPTER®(imazaquin) and ARSENAL® (imazapyr). Examples of sulfonylurea herbicidesare chlorsulfuron, metsulfuron methyl, sulfometuron methyl, chlorimuronethyl, thifensulfuron methyl, tribenuron methyl, bensulfuron methyl,nicosulfuron, ethametsulfuron methyl, rimsulfuron, triflusulfuronmethyl, triasulfuron, primisulfuron methyl, cinosulfuron, amidosulfiuon,fluzasulfuron, imazosulfuron, pyrazosulfuron ethyl and halosulfuron.

Due to their high effectiveness and low-toxicity, imidazolinoneherbicides are favored for application by spraying over the top of awide area of vegetation. The ability to spray an herbicide over the topof a wide range of vegetation decreases the costs associated withplantation establishment and maintenance, and decreases the need forsite preparation prior to use of such chemicals. Spraying over the topof a desired tolerant species also results in the ability to achievemaximum yield potential of the desired species due to the absence ofcompetitive species. However, the ability to use such spray-overtechniques is dependent upon the presence of imidazolinone-resistantspecies of the desired vegetation in the spray over area.

Among the major agricultural crops, some leguminous species such assoybean are naturally resistant to imidazolinone herbicides due to theirability to rapidly metabolize the herbicide compounds (Shaner andRobinson (1985) Weed Sci. 33:469-471). Other crops such as corn(Newhouse et al. (1992) Plant Physiol. 100:882-886) and rice (Barrett etal. (1989) Crop Safeners for Herbicides, Academic Press, New York, pp.195-220) are somewhat susceptible to imidazolinone herbicides. Thedifferential sensitivity to the imidazolinone herbicides is dependent onthe chemical nature of the particular herbicide and differentialmetabolism of the compound from a toxic to a non-toxic form in eachplant (Shaner et al. (1984) Plant Physiol. 76:545-546; Brown et al.,(1987) Pestic. Biochem. Physiol. 27:24-29). Other plant physiologicaldifferences such as absorption and translocation also play an importantrole in sensitivity (Shaner and Robinson (1985) Weed Sci. 33:469-471).

Plants resistant to imidazolinones, sulfonylureas andtriazolopyrimidines have been successfully produced using seed,microspore, pollen, and callus mutagenesis in Zea mays, Arabidopsisthaliana, Brassica napus (i.e., canola) Glycine max, Nicotiana tabacum,and Oryza sativa (Sebastian et al. (1989) Crop Sci. 29:1403-1408;Swanson et al., 1989 Theor. Appl. Genet. 78:525-530; Newhouse et al.(1991) Theor. Appl. Genet. 83:65-70; Sathasivan et al. (1991) PlantPhysiol. 97:1044-1050; Mourand et al. (1993) J. Heredity 84:91-96; U.S.Pat. No. 5,545,822). In all cases, a single, partially dominant nucleargene conferred resistance. Four imidazolinone resistant wheat plantswere also previously isolated following seed mutagenesis of Triticumaestivum L. cv. Fidel (Newhouse et al. (1992) Plant Physiol.100:882-886). Inheritance studies confirmed that a single, partiallydominant gene conferred resistance. Based on allelic studies, theauthors concluded that the mutations in the four identified lines werelocated at the same locus. One of the Fidel cultivar resistance geneswas designated FS-4 (Newhouse et al. (1992) Plant Physiol. 100:882-886).

Computer-based modeling of the three dimensional conformation of theAHAS-inhibitor complex predicts several amino acids in the proposedinhibitor binding pocket as sites where induced mutations would likelyconfer selective resistance to imidazolinones (Ott et al. (1996) J. Mol.Biol. 263:359-368). Wheat plants produced with some of these rationallydesigned mutations in the proposed binding sites of the AHAS enzyme havein fact exhibited specific resistance to a single class of herbicides(Ott et al. (1996) J. Mol. Biol. 263:359-368). Plant resistance toimidazolinone herbicides has also been reported in a number of patents.U.S. Pat. Nos. 4,761,373, 5,331,107, 5,304,732, 6,211,438, 6,211,439 and6,222,100 generally describe the use of an altered AHAS gene to elicitherbicide resistance in plants, and specifically discloses certainimidazolinone resistant corn lines. U.S. Pat. No. 5,013,659 disclosesplants exhibiting herbicide resistance due to mutations in at least oneamino acid in one or more conserved regions. The mutations describedtherein encode either cross-resistance for imidazolinones andsulfonylureas or sulfonylurea-specific resistance, butimidazolinone-specific resistance is not described. U.S. Pat. No.5,731,180 and U.S. Pat. No. 5,767,361 discuss an isolated gene having asingle amino acid substitution in a wild-type monocot AHAS amino acidsequence that results in imidazolinone-specific resistance. In addition,rice plants that are resistant to herbicides that interfere with AHAShave been developed by mutation breeding and also by the selection ofherbicide resistant plants from a pool of rice plants produced by antherculture. See, U.S. Pat. Nos. 5,545,822, 5,736,629, 5,773,703, 5,773,704,5,952,553 and 6,274,796.

In plants, as in all other organisms examined, the AHAS enzyme iscomprised of two subunits: a large subunit (catalytic role) and a smallsubunit (regulatory role) (Duggleby and Pang (2000) J. Biochem. Mol.Biol. 33:1-36). The AHAS large subunit (also referred to herein asAHASL) may be encoded by a single gene as in the case of Arabidopsis andrice or by multiple gene family members as in maize, canola, and cotton.Specific, single-nucleotide substitutions in the large subunit conferupon the enzyme a degree of insensitivity to one or more classes ofherbicides (Chang and Duggleby (1998) Biochem J. 333:765-777).

For example, bread wheat, Triticum aestivum L., contains threehomoeologous acetohydroxyacid synthase large subunit genes. Each of thegenes exhibit significant expression based on herbicide response andbiochemical data from mutants in each of the three genes (Ascenzi et al.(2003) International Society of Plant Molecular Biologists Congress,Barcelona, Spain, Ref. No. S10-17). The coding sequences of all threegenes share extensive homology at the nucleotide level (WO 03/014357).Through sequencing the AHASL genes from several varieties of Triticumaestivum, the molecular basis of herbicide tolerance in mostIMI-tolerant (imidazolinone-tolerant) lines was found to be the mutationS653(At)N, indicating a serine to asparagine substitution at a positionequivalent to the serine at amino acid 653 in Arabidopsis thaliana (WO03/01436; WO 03/014357). This mutation is due to a single nucleotidepolymorphism (SNP) in the DNA sequence encoding the AHASL protein.

Given their high effectiveness and low-toxicity, imidazolinoneherbicides are favored for agricultural use. However, the ability to useimidazolinone herbicides in a particular crop production system dependsupon the availability of imidazolinone-resistant varieties of the cropplant of interest. To produce such imidazolinone-resistant varieties,plant breeders need to develop breeding lines with theimidazolinone-resistance trait. Thus, additional imidazolinone-resistantbreeding lines and varieties of crop plants, as well as methods andcompositions for the production and use of imidazolinone-resistantbreeding lines and varieties, are needed.

SUMMARY OF THE INVENTION

The present invention provides rice plants having increased resistanceto herbicides when compared to a wild-type rice plant. In particular,the rice plants of the invention have increased resistance toimidazolinone and sulfonylurea herbicides, when compared to a wild-typerice plant. The herbicide resistant rice plants of the inventioncomprise at least one copy of a gene or polynucleotide that encodes aherbicide-resistant acetohydroxyacid synthase large subunit 1 (AHASL1)that comprise an amino acid substitution relative to the amino acidsequence of a wild-type, rice AHASL1 protein. In one embodiment of theinvention, the herbicide-resistant rice plants comprise aherbicide-resistant AHASL1 protein that comprises a valine or aspartateat amino acid position 179 or equivalent position. Theherbicide-resistant rice plant of the invention can contain one, two,three, four, or more copies of a gene or polynucleotide encoding aherbicide-resistant AHASL1 protein of the invention. The rice plants ofthe invention also include seeds and progeny plants that comprise atleast one copy of a gene or polynucleotide encoding aherbicide-resistant AHASL1 of the invention.

The present invention provides herbicide-resistant rice plants that arefrom the rice line that has been designated as IMINTA 16. A sample ofseeds of the IMINTA 16 line has been deposited with the patentdepository at NCIMB Ltd. and assigned NCIMB Accession Number NCIMB41262. The IMINTA 16 rice plants comprise in their genomes an AHASL1gene that comprises the nucleotide sequences set forth in SEQ ID NOS: 1and 3, or that encodes the AHASL1 protein comprising, the amino acidsequence set forth in SEQ ID NO: 2. When compared to the amino acidsequence of the AHASL1 protein that is encoded by an AHASL1 gene from awild-type rice plant (GenBank Accession No. AB049822), the amino acidsequence set forth in SEQ ID NO: 2 possesses a single amino aciddifference from the wild-type amino acid sequence. In the amino acidsequence set forth in SEQ ID NO: 2, there is a valine at amino acidposition 179. In the amino acid sequence of the wild-type, rice AHASL1protein, this same amino acid position has an alanine.

The present invention further provides isolated polynucleotides andisolated polypeptides for rice (Oryza sativa) AHASL1 proteins. Thepolynucleotides of the invention encompass nucleotide sequences thatencode herbicide-resistant AHASL1 proteins. The herbicide-resistantAHASL1 proteins of the invention are imidazolinone-resistant AHASL1proteins that comprise an alanine-to-valine substitution at position 179in their respective amino acid sequences, when compared to thecorresponding wild-type amino acid sequence. The polynucleotides of theinvention encompass the nucleotide sequences set forth in SEQ ID NOS: 1and 3, nucleotide sequences encoding the amino acid sequence set forthin SEQ ID NO: 2, and fragments and variants of said nucleotide sequencesthat encode proteins comprising AHAS activity, particularlyherbicide-resistant AHAS activity.

The present invention provides expression cassettes for expressing thepolynucleotides of the invention in plants, plant cells, and othernon-human host cells. The expression cassettes comprise a promoterexpressible in the plant, plant cell, or other host cells of interestoperably linked to a polynucleotide of the invention that encodes aherbicide-resistant AHASL1 protein. If necessary for targetingexpression to the chloroplast, the expression cassette can also comprisean operably linked chloroplast-targeting sequence that encodes of achloroplast transit peptide to direct an expressed AHASL1 protein to thechloroplast. The expression cassettes of the invention find use in amethod for enhancing the herbicide tolerance of a plant and a host cell.The method involves transforming the plant or host cell with anexpression cassette of the invention, wherein the expression cassettecomprises a promoter that is expressible in the plant or host cell ofinterest and the promoter is operably linked to a polynucleotide of theinvention that encodes an herbicide-resistant AHASL1 protein of theinvention. The method further comprises regenerating a transformed plantfrom the transformed plant cell.

The present invention provides a method for increasing AHAS activity ina plant comprising transforming a plant cell with a polynucleotideconstruct comprising a nucleotide sequence operably linked to a promoterthat drives expression in a plant cell and regenerating a transformedplant from the transformed plant cell. The nucleotide sequence isselected from those nucleotide sequences that encode theherbicide-resistant AHASL1 proteins of the invention, particularly thenucleotide sequences set forth in SEQ ID NOS: 1 and 3, nucleotidesequences encoding the amino acid sequence set forth in SEQ ID NO: 2,and fragments and variants thereof. A plant produced by this methodcomprises increased AHAS activity, when compared to an untransformedplant.

The present invention provides a method for producing aherbicide-resistant plant comprising transforming a plant cell with apolynucleotide construct comprising a nucleotide sequence operablylinked to a promoter that drives expression in a plant cell andregenerating a transformed plant from said transformed plant cell. Thenucleotide sequence is selected from those nucleotide sequences thatencode the herbicide-resistant AHASL1 proteins of the invention,particularly the nucleotide sequences set forth in SEQ ID NOS: 1 and 3,nucleotide sequences encoding the amino acid sequence set forth in SEQID NO: 2, and fragments and variants thereof, including, but not limitedto, the mature forms of the herbicide-resistant AHASL1 proteins of theinvention. A herbicide-resistant plant produced by this method comprisesenhanced resistance to at least one herbicide, particularly animidazolinone or sulfonylurea herbicide, when compared to anuntransformed plant.

The present invention provides a method for enhancingherbicide-tolerance in a herbicide-tolerant plant. The method finds usein enhancing the resistance of a plant that already is resistant to alevel of a herbicide that would kill or significantly injure a wild-typeplant. Such a herbicide-tolerant plant can be a herbicide-tolerant plantthat has been genetically engineered for herbicide-tolerance or aherbicide-tolerant plant that was developed by means that do not involverecombinant DNA such as, for example, the IMINTA 16 rice plants of thepresent invention. The method comprises transforming aherbicide-tolerant plant with a polynucleotide construct comprising anucleotide sequence operably linked to a promoter that drives expressionin a plant cell and regenerating a transformed plant from thetransformed plant cell. The nucleotide sequence is selected from thosenucleotide sequences that encode the herbicide-resistant AHASL1 proteinsof the invention, particularly the nucleotide sequences set forth in SEQID NO: 1 and 3, nucleotide sequences encoding the amino acid sequenceset forth in SEQ ID NO: 2, and fragments and variants thereof.

The present invention provides transformation vectors comprising aselectable marker gene of the invention. The selectable marker genecomprises a promoter that drives expression in a host cell operablylinked to a polynucleotide comprising a nucleotide sequence that encodesa herbicide-resistant AHASL1 protein of the invention. Thetransformation vector can additionally comprise a gene of interest to beexpressed in the host cell and can also, if desired, include achloroplast-targeting sequence that is operably linked to thepolynucleotide of the invention.

The present invention further provides methods for using thetransformation vectors of the invention to select for cells transformedwith the gene of interest. Such methods involve the transformation of ahost cell with the transformation vector, exposing the cell to a levelof an imidazolinone or sulfonylurea herbicide that would kill or inhibitthe growth of a non-transformed host cell, and identifying thetransformed host cell by its ability to grow in the presence of theherbicide. In one embodiment of the invention, the host cell is a plantcell and the selectable marker gene comprises a promoter that drivesexpression in a plant cell.

The present invention provides a method for controlling weeds in thevicinity of the herbicide-resistant plants of the invention, includingthe herbicide-resistant rice plants described above and plantstransformed with the herbicide-resistant AHASL1 polynucleotides of theinvention. Such transformed plants comprise in their genomes at leastone expression cassette comprising a promoter that drives geneexpression in a plant cell, wherein the promoter is operably linked toan AHASL1 polynucleotide of the invention. The method comprises applyingan effective amount of a herbicide to the weeds and to theherbicide-resistant plant, wherein the herbicide-resistant plant, planthas increased resistance to at least one herbicide, particularly animidazolinone or sulfonylurea herbicide, when compared to a wild-type oruntransformed plant.

The plants of the present invention can be transgenic or non-transgenic.An example of a non-transgenic rice plant having increased resistance toimidazolinone and/or sulfonylurea herbicides includes a rice planthaving NCIMB Accession Number NCIMB 41262, or mutant, recombinant, or agenetically engineered derivative of the plant having NCIMB AccessionNumber NCIMB 41262; or of any progeny of the plant having NCIMBAccession Number NCIMB 41262; or a plant that is a progeny of any ofthese plants; or a plant that comprises the herbicide resistancecharacteristics of the plant having NCIMB Accession Number NCIMB 41262.

The present invention also provides plants, plant organs, plant tissues,plant cells, seeds, and non-human host cells that are transformed withthe at least one polynucleotide, expression cassette, or transformationvector of the invention. Such transformed plants, plant organs, planttissues, plant cells, seeds, and non-human host cells have enhancedtolerance or resistance to at least one herbicide, at levels of theherbicide that kill or inhibit the growth of an untransformed plant,plant tissue, plant cell, or non-human host cell, respectively.Preferably, the transformed plants, plant tissues, plant cells, andseeds of the invention are Arabidopsis thaliana and crop plants.

BRIEF DESCRIPTION OF THE DRAWING(S)

FIG. 1 is a nucleotide sequence alignment of the herbicide-resistantrice AHASL1 gene of the present invention (SEQ ID NO: 1) with some knownplant AHASL nucleotide sequences. The C-to-T transition (relative towild-type) at nucleotide 542 in SEQ ID NO: 1 is indicated by white typewithin a black box. The start and stop codons are represented inbold-face type. The names of the other AHASL sequences in the figure aredefined as follows: “OsAHASL1.1” is a wild-type AHASL1 nucleotidesequence from El Paso rice background; “OsAHASL1.2” is a wild-typeAHASL1 nucleotide sequence from IRGA rice background; “OsAHASL1.4” is arice AHASL1 nucleotide sequence (Accession No. AB049822); “OsAHASL1.6”is a rice AHASL1 nucleotide sequence (Accession No. AB049823);“ZmAHASL1” is a corn AHASL1 nucleotide sequence (Accession No. X63554);“ZmAHASL2” is a corn AHASL2 nucleotide sequence (Accession No. X63553);“OsAHASL2” is a rice AHASL2 nucleotide sequence (Accession No.AL731599); “AtAHASL” is an Arabidopsis thaliana AHASL nucleotidesequence (Accession No. AY124092).

FIG. 2 is an amino acid sequence alignment of the herbicide-resistantrice AHASL1 protein of the present invention (SEQ ID NO: 2) with someknown plant AHASL nucleotide sequences. The Ala-to-Val substitution(relative to wild-type) at position 179 in SEQ ID NO: 2 is indicated bywhite type within a black box. The initial methionine (M) is representedin bold-face type. The other names used in the figure refer to the aminoacid sequences encoded by the nucleotide sequences indicated in thedescription of FIG. 1 above.

SEQUENCE LISTING

The nucleotide and amino acid sequences listed in the accompanyingsequence listing are shown using standard letter abbreviations fornucleotide bases, and three-letter code for amino acids. The nucleotidesequences follow the standard convention of beginning at the 5′ end ofthe sequence and proceeding forward (i.e., from left to right in eachline) to the 3′ end. Only one strand of each nucleic acid sequence isshown, but the complementary strand is understood to be included by anyreference to the displayed strand. The amino acid sequences follow thestandard convention of beginning at the amino terminus of the sequenceand proceeding forward (i.e., from left to right in each line) to thecarboxyl terminus.

SEQ ID NO: 1 sets forth the nucleotide sequence encoding animidazolinone-resistant AHASL1 protein from rice with the Ala₁₇₉-to-Valsubstitution. The coding region of SEQ ID NO: 1 corresponds tonucleotides 7 to 1938.

SEQ ID NO: 2 sets forth the amino acid sequence of animidazolinone-resistant AHASL1 protein from rice with the Ala₁₇₉-to-Valsubstitution that is encoded by the nucleotide sequence set forth in SEQID NO: 1.

SEQ ID NO: 3 sets forth the nucleotide sequence of the coding region ofSEQ ID NO: 1.

SEQ ID NOS: 4-18 set forth the nucleotide sequences of primers used forthe PCR amplification and DNA sequencing of the AHASL1 gene of theinvention as described in Example 2 below (see, Table 1).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to rice plants having increased resistanceto herbicides when compared to a wild-type rice plant. The herbicideresistant rice plants of the invention were produced as describedhereinbelow by exposing wild-type (with respect to herbicide resistance)rice seeds to a mutagen, sowing the seeds, allowing the plants to matureand reproduce, and selecting progeny plants that displayed enhancedresistance to imidazolinone herbicides, relative to the resistance of awild-type rice plant. The invention provides the herbicide resistantrice line and plants thereof that are referred to herein IMINTA 16. Suchherbicide resistant rice plants find use in methods for controllingweeds, particularly red rice and other weeds that are sensitive toimidazolinone and sulfonylurea herbicides.

From the IMINTA 16 herbicide-resistant rice plants, the coding region ofan acetohydroxyacid synthase large subunit 1 (AHASL1) gene was isolatedby polymerase chain reaction (PCR) amplification and sequenced. Bycomparing the polynucleotide sequences of the herbicide resistant riceplants of the invention to a rice AHASL1 cDNA from a wild-type riceplant (GenBank Accession No. AB049822), it was discovered that thecoding region of the AHASL1 polynucleotide sequence from IMINTA 16differed from the wild-type rice AHASL1 cDNA sequence by a singlenucleotide. For the AHASL1 polynucleotide sequence of IMINTA 16, therewas a C-to-T transition at nucleotide 542 (SEQ ID NO: 1, FIG. 1). ThisC-to-T transition in the AHASL1 polynucleotide sequence results in aAla-to-Val substitution at amino acid 179 in a conserved region of thepredicted amino acid sequence of the AHASL1 protein, relative to theamino acid sequence of the wild-type AHASL1 protein (FIG. 2).

The invention further relates to isolated polynucleotide moleculescomprising nucleotide sequences that encode the herbicide-resistantAHASL1 proteins of IMINTA 16 rice plants and to such AHASL1 proteins.The invention discloses the isolation and nucleotide sequence of apolynucleotide encoding a herbicide-resistant rice AHASL1 protein fromherbicide-resistant rice plant that was produced by chemical mutagenesisof wild-type rice plants. The herbicide-resistant AHASL1 proteins of theinvention comprise an alanine-to-valine substitution at position 179 intheir respective amino acid sequences, when compared to thecorresponding wild-type AHASL1 amino acid sequence.

The present invention provides isolated polynucleotide molecules thatencode herbicide resistant AHASL1 proteins from rice (Oryza sativa L.).Specifically, the invention provides isolated polynucleotide moleculescomprising: the nucleotide sequences set forth in SEQ ID NOS: 1 and 3,nucleotide sequences encoding AHASL1 proteins comprising the amino acidsequence set forth in SEQ ID NO: 2, and fragments and variants of suchnucleotide sequences that encode functional AHASL1 proteins thatcomprise herbicide-resistant AHAS activity.

The isolated herbicide-resistant AHASL1 polynucleotide molecules of theinvention comprise nucleotide sequences that encode herbicide-resistantAHASL1 proteins. Such polynucleotide molecules can be used inpolynucleotide constructs for the transformation of plants, particularlycrop plants, to enhance the resistance of the plants to herbicides,particularly herbicides that are known to inhibit AHAS activity, moreparticularly imidazolinone and sulfonylurea herbicides. Suchpolynucleotide constructs can be used in expression cassettes,expression vectors, transformation vectors, plasmids and the like. Thetransgenic plants obtained following transformation with suchpolynucleotide constructs show increased resistance to AHAS-inhibitingherbicides such as, for example, imidazolinone and sulfonylureaherbicides.

Compositions of the invention include polynucleotide moleculescomprising nucleotide sequences that encode AHASL1 proteins. Inparticular, the present invention provides for isolated polynucleotidemolecules comprising nucleotide sequences encoding the amino acidsequence shown in SEQ ID NO: 2, and fragments and variants thereof thatencode polypeptides comprising AHAS activity. Further provided arepolypeptides having an amino acid sequence encoded by a polynucleotidemolecule described herein, for example those set forth in SEQ ID NOS: 1and 3, and fragments and variants thereof that encode polypeptidescomprising AHAS activity, particularly herbicide-resistant AHASLactivity. The polynucleotides molecules of the invention furtherencompass nucleotide sequences that encode mature forms of the AHASL1proteins described above. Such mature forms of AHASL1 proteins compriseAHAS activity, particularly herbicide-resistant AHAS activity, but lackthe chloroplast transit peptide that is part of full-length AHASL1proteins.

The invention encompasses isolated or substantially purified nucleicacid or protein compositions. An “isolated” or “purified” polynucleotidemolecule or protein, or biologically active portion thereof, issubstantially or essentially free from components that normallyaccompany or interact with the polynucleotide molecule or protein asfound in its naturally occurring environment. Thus, an isolated orpurified polynucleotide molecule or protein is substantially free ofother cellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized. Preferably, an “isolated” nucleicacid is free of sequences (preferably protein encoding sequences) thatnaturally flank the nucleic acid (i.e., sequences located at the 5′ and3′ ends of the nucleic acid) in the genomic DNA of the organism fromwhich the nucleic acid is derived. For example, in various embodiments,the isolated polynucleotide molecule can contain less than about 5 kb, 4kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences thatnaturally flank the polynucleotide molecule in genomic DNA of the cellfrom which the nucleic acid is derived. A protein that is substantiallyfree of cellular material includes preparations of protein having lessthan about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminatingprotein. When the protein of the invention or biologically activeportion thereof is recombinantly produced, preferably culture mediumrepresents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) ofchemical precursors or non-protein-of-interest chemicals.

The present invention provides isolated polypeptides comprising AHASL1proteins. The isolated polypeptides comprise an amino acid sequenceselected from the group consisting of the amino acid sequence set forthin SEQ ID NO: 2, the amino acid sequences encoded by nucleotidesequences set forth in SEQ ID NOS: 1 and 3, and functional fragments andvariants of said amino acid sequences that encode an AHASL1 polypeptidecomprising AHAS activity. By “functional fragments and variants” isintended fragments and variants of the exemplified polypeptides thatcomprise AHAS activity.

Additionally provided are isolated polypeptides comprising the matureforms of the AHASL1 proteins of the invention. Such mature forms ofAHASL1 proteins comprise AHAS activity, particularly herbicide-resistantAHAS activity, but lack the chloroplast transit peptide that is part offull-length AHASL1 proteins.

In certain embodiments of the invention, the methods involve the use ofherbicide-tolerant or herbicide-resistant plants. By an“herbicide-tolerant” or “herbicide-resistant” plant, it is intended thata plant that is tolerant or resistant to at least one herbicide at alevel that would normally kill, or inhibit the growth of, a normal orwild-type plant. In one embodiment of the invention, theherbicide-tolerant plants of the invention comprise a herbicide-tolerantor herbicide-resistant AHASL1 protein. By “herbicide-tolerant AHASL1protein” or “herbicide-resistant AHASL1 protein”, it is intended thatsuch an AHASL1 protein displays higher AHAS activity, relative to theAHAS activity of a wild-type AHASL1 protein, when in the presence of atleast one herbicide that is known to interfere with AHAS activity and ata concentration or level of the herbicide that is to known to inhibitthe AHAS activity of the wild-type AHASL1 protein. Furthermore, the AHASactivity of such a herbicide-tolerant or herbicide-resistant AHASL1protein may be referred to herein as “herbicide-tolerant” or“herbicide-resistant” AHAS activity.

For the present invention, the terms “herbicide-tolerant” and“herbicide-resistant” are used interchangeable and are intended to havean equivalent meaning and an equivalent scope. Similarly, the terms“herbicide-tolerance” and “herbicide-resistance” are usedinterchangeable and are intended to have an equivalent meaning and anequivalent scope. Likewise, the terms “imidazolinone-resistant” and“imidazolinone-resistance” are used interchangeable and are intended tobe of an equivalent meaning and an equivalent scope as the terms“imidazolinone-tolerant” and “imidazolinone-tolerance”, respectively.

The invention encompasses herbicide-resistant AHASL1 polynucleotides andherbicide-resistant AHASL1 proteins. By “herbicide-resistant AHASL1polynucleotide” is intended a polynucleotide that encodes a proteincomprising herbicide-resistant AHAS activity. By “herbicide-resistantAHASL1 protein” is intended a protein or polypeptide that comprisesherbicide-resistant AHAS activity.

Further, it is recognized that a herbicide-tolerant orherbicide-resistant AHASL1 protein can be introduced into a plant bytransforming a plant or ancestor thereof with a nucleotide sequenceencoding a herbicide-tolerant or herbicide-resistant AHASL1 protein.Such herbicide-tolerant or herbicide-resistant AHASL1 proteins areencoded by the herbicide-tolerant or herbicide-resistant AHASL1polynucleotides. Alternatively, a herbicide-tolerant orherbicide-resistant AHASL1 protein may occur in a plant as a result of anaturally occurring or induced mutation in an endogenous AHASL1 gene inthe genome of a plant or progenitor thereof.

The present invention provides plants, plant tissues, plant cells, andhost cells with increased resistance or tolerance to at least oneherbicide, particularly an imidazolinone or sulfonylurea herbicide. Thepreferred amount or concentration of the herbicide is an “effectiveamount” or “effective concentration.” By “effective amount” and“effective concentration” is intended an amount and concentration,respectively, that is sufficient to kill or inhibit the growth of asimilar, wild-type, plant, plant tissue, plant cell, or host cell, butthat said amount does not kill or inhibit as severely the growth of theherbicide-resistant plants, plant tissues, plant cells, and host cellsof the present invention. Typically, the effective amount of a herbicideis an amount that is routinely used in agricultural production systemsto kill weeds of interest. Such an amount is known to those of ordinaryskill in the art.

By “similar, wild-type, plant, plant tissue, plant cell or host cell” isintended a plant, plant tissue, plant cell, or host cell, respectively,that lacks the herbicide-resistance characteristics and/or particularpolynucleotide of the invention that are disclosed herein. The use ofthe term “wild-type” is not, therefore, intended to imply that a plant,plant tissue, plant cell, or other host cell lacks recombinant DNA inits genome, and/or does not possess herbicide resistant characteristicsthat are different from those disclosed herein.

As used herein unless clearly indicated otherwise, the term “plant”intended to mean a plant any developmental stage, as well as any part orparts of a plant that may be attached to or separate from a whole intactplant. Such parts of a plant include, but are not limited to, organs,tissues, and cells of a plant. Examples of particular plant partsinclude a stem, a leaf, a root, an inflorescence, a flower, a floret, afruit, a pedicle, a peduncle, a stamen, an anther, a stigma, a style, anovary, a petal, a sepal, a carpel, a root tip, a root cap, a root hair,a leaf hair, a seed hair, a pollen grain, a microspore, a cotyledon, ahypocotyl, an epicotyl, xylem, phloem, parenchyma, endosperm, acompanion cell, a guard cell, and any other known organs, tissues, andcells of a plant. Furthermore, it is recognized that a seed is a plant.

The plants of the present invention include both non-transgenic plantsand transgenic plants. By “non-transgenic plant” is intended mean aplant lacking recombinant DNA in its genome. By “transgenic plant” isintended to mean a plant comprising recombinant DNA in its genome. Sucha transgenic plant can be produced by introducing recombinant DNA intothe genome of the plant. When such recombinant DNA is incorporated intothe genome of the transgenic plant, progeny of the plant can alsocomprise the recombinant DNA. A progeny plant that comprises at least aportion of the recombinant DNA of at least one progenitor transgenicplant is also a transgenic plant.

The present invention provides the herbicide-resistant rice line andplants thereof known as IMINTA 16. A deposit of at least 250 seeds ofIMINTA 16 was made to the patent depository of NCIMB Ltd., FergusonBuilding, Craibstone Estate Bucksburn, Aberdeen, AB21 9YA, Scotland, UKon Jan. 5, 2005 and assigned NCIMB Accession Number NCIMB 41262. Due toa shortage of seeds of the IMINTA 16 line at the time of filing, lessthan 2500 seeds of the IMINTA 16 line were submitted to NCIMB Ltd. priorto filing. Applicants will supply additional seeds of the IMINTA 16 lineto reach a total of at least 2500 seeds as the seeds become available.These deposits will be maintained under the terms of the Budapest Treatyon the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedure. The deposit of the seeds of IMINTA 16was made for a term of at least 30 years and at least 5 years after themost recent request for the furnishing of a sample of that deposit isreceived by NCIMB Ltd. Additionally, Applicants have satisfied all therequirements of 37 C.F.R. §§1.801-1.809, including providing anindication of the viability of the sample.

The present invention provides herbicide-resistant rice plants of theIMINTA 16 line that were produced by a mutation breeding. Wild-type riceplants were mutagenized by exposing the plants to a mutagen,particularly a chemical mutagen, more particularly sodium azide.However, the present invention is not limited to herbicide-resistantrice plants that are produced by a mutagensis method involving thechemical mutagen sodium azide. Any mutagensis method known in the artmay be used to produce the herbicide-resistant rice plants of thepresent invention. Such mutagensis methods can involve, for example, theuse of any one or more of the following mutagens: radiation, such asX-rays, Gamma rays (e.g., cobalt 60 or cesium 137), neutrons, (e.g.,product of nuclear fission by uranium 235 in an atomic reactor), Betaradiation (e.g., emitted from radioisotopes such as phosphorus 32 orcarbon 14), and ultraviolet radiation (preferably from 2500 to 2900 nm),and chemical mutagens such as base analogues (e.g., 5-bromo-uracil),related compounds (e.g., 8-ethoxy caffeine), antibiotics (e.g.,streptonigrin), alkylating agents (e.g., sulfur mustards, nitrogenmustards, epoxides, ethylenamines, sulfates, sulfonates, sulfones,lactones), azide, hydroxylamine, nitrous acid, ethyl methanesulfonate(EMS), or acridines. Herbicide-resistant plants can also be produced byusing tissue culture methods to select for plant cells comprisingherbicide-resistance mutations and then regenerating herbicide-resistantplants therefrom. See, for example, U.S. Pat. Nos. 5,773,702 and5,859,348, both of which are herein incorporated in their entirety byreference. Further details of mutation breeding can be found in“Principals of Cultivar Development” Fehr, 1993 Macmillan PublishingCompany the disclosure of which is incorporated herein by reference.

Analysis of the AHASL1 gene of the rice plants of IMINTA 16 linerevealed a point mutation. In the AHASL1 gene from IMINTA 16, the pointmutation results in the substitution of a valine for the alanine that isfound at amino acid position 179 in the wild-type AHASL1 amino acidsequence of GenBank Accession No. AB049822. Thus, the present inventiondiscloses that substituting another amino acid for the alanine at aminoacid position 179 of the rice AHASL1 protein can cause a rice plant tohave enhanced resistance to a herbicide, particularly an imidazolinoneand/or sulfonylurea herbicide. As disclosed in Example 3 below, alanine179 is found in a conserved region of AHASL proteins and other aminoacid substitutions for the alanine at 179 have been disclosed that areknown to confer herbicide resistance on a plant that comprises such anAHASL protein. Accordingly, the herbicide-resistant rice plants of theinvention include, but are not limited to those rice plants whichcomprise in their genomes at least one copy of an AHASL1 polynucleotidethat encodes a herbicide-resistant AHASL1 protein that comprises anaspartate or valine at amino acid position 179 or equivalent position.

The rice plants of the invention additionally include plants thatcomprise, relative to the wild-type AHASL1 protein, an aspartate orvaline at amino acid position 179 or equivalent position and one or moreadditional amino acid substitutions in the AHASL1 protein relative tothe wild-type AHASL1 protein, wherein such a rice plant has increasedresistance to at least one herbicide when compared to a wild-type riceplant. Such additional amino acid substitutions include, but are notlimited to: a threonine at amino acid position 96 or equivalentposition; an alanine, threonine, histidine, leucine, arginine,isoleucine, glutamine, or serine at amino acid position 171 orequivalent position; a leucine at amino acid position 548 or equivalentposition; and an asparagine, threonine, or phenylalanine at amino acidposition 627 or equivalent position.

The present invention provides AHASL1 proteins with amino acidsubstitutions at particular amino acid positions within conservedregions of the rice AHASL1 proteins disclosed herein. Unless otherwiseindicated herein, particular amino acid positions refer to the positionof that amino acid in the full-length rice AHASL1 amino acid sequencesset forth in SEQ ID NO: 2. Furthermore, those of ordinary skill in theart will recognize that such amino acid positions can vary depending onwhether amino acids are added or removed from, for example, theN-terminal end of an amino acid sequence. Thus, the inventionencompasses the amino substitutions at the recited position orequivalent position (e.g., “amino acid position 179 or equivalentposition”). By “equivalent position” is intended to mean a position thatis within the same conserved region as the exemplified amino acidposition. Examples of such conserved regions are provided in Table 2below.

In addition, the present invention provides rice AHASL1 polypeptidescomprising an aspartate or valine at amino acid position 179 orequivalent position and one or more additional amino acid substitutionsin the AHASL1 protein, relative to the wild-type AHASL1 protein, whereinAHASL1 polypeptide comprises herbicide tolerant AHAS activity, whencompared to the AHAS activity of a wild-type AHASL1. These amino acidsubstitutions include, but are not limited, to those that are known toconfer resistance on a plant to at least one herbicide, particularly animidazolinone herbicide and/or a sulfonylurea herbicide. Such additionalamino acid substitutions include, but are not limited to: a threonine atamino acid position 96 or equivalent position; an alanine, threonine,histidine, leucine, arginine, isoleucine, glutamine, or serine at aminoacid position 171 or equivalent position; a leucine at amino acidposition 548 or equivalent position; and an asparagine, threonine, orphenylalanine at amino acid position 627 or equivalent position. Theinvention further provides isolated polynucleotides encoding such AHASL1polypeptides, as well as expression cassettes, transformation vectors,transformed host cells, transformed plants, and methods comprising suchpolynucleotides.

The present invention provides methods for enhancing the tolerance orresistance of a plant, plant tissue, plant cell, or other host cell toat least one herbicide that interferes with the activity of the AHASenzyme. Preferably, such an AHAS-inhibiting herbicide is animidazolinone herbicide, a sulfonylurea herbicide, a triazolopyrimidineherbicide, a pyrimidinyloxybenzoate herbicide, asulfonylamino-carbonyltriazolinone herbicide, or mixture thereof. Morepreferably, such a herbicide is an imidazolinone herbicide, asulfonylurea herbicide, or mixture thereof. For the present invention,the imidazolinone herbicides include, but are not limited to, PURSUIT®(imazethapyr), CADRE® (imazapic), RAPTOR® (imazamox), SCEPTER®(imazaquin), ASSERT® (imazethabenz), ARSENAL® (imazapyr), a derivativeof any of the aforementioned herbicides, and a mixture of two or more ofthe aforementioned herbicides, for example, imazapyr/imazamox(ODYSSEY®). More specifically, the imidazolinone herbicide can beselected from, but is not limited to,2-(4-isopropyl-4-methyl-5-oxo-2-imidiazolin-2-yl)-nicotinic acid,[2-(4-isopropyl)-4-][methyl-5-oxo-2-imidazolin-2-yl)-3-quinolinecarboxylic] acid,[5-ethyl-2-(4-isopropyl-] 4-methyl-5-oxo-2-imidazolin-2-yl)-nicotinicacid,2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-5-(methoxymethyl)-nicotinicacid, [2-(4-isopropyl-4-methyl-5-oxo-2-]imidazolin-2-yl)-5-methylnicotinic acid, and a mixture of methyl[6-(4-isopropyl-4-] methyl-5-oxo-2-imidazolin-2-yl)-m-toluate and methyl[2-(4-isopropyl-4-methyl-5-] oxo-2-imidazolin-2-yl)-p-toluate. The useof 5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-nicotinicacid and [2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-]yl)-5-(methoxymethyl)-nicotinic acid is preferred. The use of[2-(4-isopropyl-4-]methyl-5-oxo-2-imidazolin-2-yl)-5-(methoxymethyl)-nicotinic acid isparticularly preferred.

For the present invention, the sulfonylurea herbicides include, but arenot limited to, chlorsulfuron, metsulfuron methyl, sulfometuron methyl,chlorimuron ethyl, thifensulfuron methyl, tribenuron methyl, bensulfuronmethyl, nicosulfuron, ethametsulfuron methyl, rimsulfuron,triflusulfuron methyl, triasulfuron, primisulfuron methyl, cinosulfuron,amidosulfiuon, fluzasulfuron, imazosulfuron, pyrazosulfuron ethyl,halosulfuron, azimsulfuron, cyclosulfuron, ethoxysulfuron,flazasulfuron, flupyrsulfuron methyl, foramsulfuron, iodosulfuron,oxasulfuron, mesosulfuron, prosulfuron, sulfosulfuron, trifloxysulfuron,tritosulfuron, a derivative of any of the aforementioned herbicides, anda mixture of two or more of the aforementioned herbicides. Thetriazolopyrimidine herbicides of the invention include, but are notlimited to, cloransulam, diclosulam, florasulam, flumetsulam, metosulam,and penoxsulam. The pyrimidinyloxybenzoate herbicides of the inventioninclude, but are not limited to, bispyribac, pyrithiobac, pyriminobac,pyribenzoxim and pyriftalid. The sulfonylamino-carbonyltriazolinoneherbicides include, but are not limited to, flucarbazone andpropoxycarbazone.

It is recognized that pyrimidinyloxybenzoate herbicides are closelyrelated to the pyrimidinylthiobenzoate herbicides and are generalizedunder the heading of the latter name by the Weed Science Society ofAmerica. Accordingly, the herbicides of the present invention furtherinclude pyrimidinylthiobenzoate herbicides, including, but not limitedto, the pyrimidinyloxybenzoate herbicides described above.

The present invention provides methods for enhancing AHAS activity in aplant comprising transforming a plant with a polynucleotide constructcomprising a promoter operably linked to an AHASL1 nucleotide sequenceof the invention. The methods involve introducing a polynucleotideconstruct of the invention into at least one plant cell and regeneratinga transformed plant therefrom. The methods involve the use of a promoterthat is capable of driving gene expression in a plant cell. Preferably,such a promoter is a constitutive promoter or a tissue-preferredpromoter. The methods find use in enhancing or increasing the resistanceof a plant to at least one herbicide that interferes with the catalyticactivity of the AHAS enzyme, particularly an imidazolinone orsulfonylurea herbicide.

The present invention provides expression cassettes for expressing thepolynucleotides of the invention in plants, plant tissues, plant cells,and other host cells. The expression cassettes comprise a promoterexpressible in the plant, plant tissue, plant cell, or other host cellsof interest operably linked to a polynucleotide of the invention thatcomprises a nucleotide sequence encoding either a full-length (i.e.including the chloroplast transit peptide) or mature AHASL1 protein(i.e. without the chloroplast transit peptide). If expression is desiredin the plastids or chloroplasts of plants or plant cells, the expressioncassette may also comprise an operably linked chloroplast-targetingsequence that encodes a chloroplast transit peptide.

The expression cassettes of the invention find use in a method forenhancing the herbicide tolerance of a plant or a host cell. The methodinvolves transforming the plant or host cell with an expression cassetteof the invention, wherein the expression cassette comprises a promoterthat is expressible in the plant or host cell of interest and thepromoter is operably linked to a polynucleotide of the invention thatcomprises a nucleotide sequence encoding an imidazolinone-resistantAHASL1 protein of the invention.

The use of the term “polynucleotide constructs” herein is not intendedto limit the present invention to polynucleotide constructs comprisingDNA. Those of ordinary skill in the art will recognize thatpolynucleotide constructs, particularly polynucleotides andoligonucleotides, comprised of ribonucleotides and combinations ofribonucleotides and deoxyribonucleotides may also be employed in themethods disclosed herein. Thus, the polynucleotide constructs of thepresent invention encompass all polynucleotide constructs that can beemployed in the methods of the present invention for transforming plantsincluding, but not limited to, those comprised of deoxyribonucleotides,ribonucleotides, and combinations thereof. Such deoxyribonucleotides andribonucleotides include both naturally occurring molecules and syntheticanalogues. The polynucleotide constructs of the invention also encompassall forms of polynucleotide constructs including, but not limited to,single-stranded forms, double-stranded forms, hairpins, stem-and-loopstructures, and the like. Furthermore, it is understood by those ofordinary skill the art that each nucleotide sequences disclosed hereinalso encompasses the complement of that exemplified nucleotide sequence.

Furthermore, it is recognized that the methods of the invention mayemploy a polynucleotide construct that is capable of directing, in atransformed plant, the expression of at least one protein, or at leastone RNA, such as, for example, an antisense RNA that is complementary toat least a portion of an mRNA. Typically such a polynucleotide constructis comprised of a coding sequence for a protein or RNA operably linkedto 5′ and 3′ transcriptional regulatory regions. Alternatively, it isalso recognized that the methods of the invention may employ apolynucleotide construct that is not capable of directing, in atransformed plant, the expression of a protein or RNA.

Further, it is recognized that, for expression of a polynucleotides ofthe invention in a host cell of interest, the polynucleotide istypically operably linked to a promoter that is capable of driving geneexpression in the host cell of interest. The methods of the inventionfor expressing the polynucleotides in host cells do not depend onparticular promoter. The methods encompass the use of any promoter thatis known in the art and that is capable of driving gene expression inthe host cell of interest.

The present invention encompasses AHASL1 polynucleotide molecules andfragments and variants thereof. Polynucleotide molecules that arefragments of these nucleotide sequences are also encompassed by thepresent invention. By “fragment” is intended a portion of the nucleotidesequence encoding an AHASL1 protein of the invention. A fragment of anAHASL1 nucleotide sequence of the invention may encode a biologicallyactive portion of an AHASL1 protein, or it may be a fragment that can beused as a hybridization probe or PCR primer using methods disclosedbelow. A biologically active portion of an AHASL1 protein can beprepared by isolating a portion of one of the AHASL1 nucleotidesequences of the invention, expressing the encoded portion of the AHASL1protein (e.g., by recombinant expression in vitro), and assessing theactivity of the encoded portion of the AHASL1 protein. Polynucleotidemolecules that are fragments of an AHASL1 nucleotide sequence compriseat least about 15, 20, 50, 75, 100, 200, 300, 350, 400, 450, 500, 550,600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200,1250, 1300, 1350, 1400, 1500, 1600, 1700, 1800, 1900, or 1950nucleotides, or up to the number of nucleotides present in a full-lengthnucleotide sequence disclosed herein (for example, 1961 and 1932nucleotides for SEQ ID NOS: 1 and 3, respectively) depending upon theintended use.

A fragment of an AHASL1 nucleotide sequence that encodes a biologicallyactive portion of an AHASL1 protein of the invention will encode atleast about 15, 25, 30, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350,400, 450, 500, 550, 600, or 625 contiguous amino acids, or up to thetotal number of amino acids present in a full-length AHASL1 protein ofthe invention (for example, 644 amino acids for SEQ ID NO: 2). Fragmentsof an AHASL1 nucleotide sequence that are useful as hybridization probesfor PCR primers generally need not encode a biologically active portionof an AHASL1 protein.

Polynucleotide molecules that are variants of the nucleotide sequencesdisclosed herein are also encompassed by the present invention.“Variants” of the AHASL1 nucleotide sequences of the invention includethose sequences that encode the AHASL1 proteins disclosed herein butthat differ conservatively because of the degeneracy of the geneticcode. These naturally occurring allelic variants can be identified withthe use of well-known molecular biology techniques, such as polymerasechain reaction (PCR) and hybridization techniques as outlined below.Variant nucleotide sequences also include synthetically derivednucleotide sequences that have been generated, for example, by usingsite-directed mutagenesis but which still encode the AHASL1 proteindisclosed in the present invention as discussed below. Generally,nucleotide sequence variants of the invention will have at least about90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to aparticular nucleotide sequence disclosed herein. A variant AHASL1nucleotide sequence will encode an AHASL1 protein, respectively, thathas an amino acid sequence having at least about 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence ofan AHASL1 protein disclosed herein.

In addition, the skilled artisan will further appreciate that changescan be introduced by mutation into the nucleotide sequences of theinvention thereby leading to changes in the amino acid sequence of theencoded AHASL1 proteins without altering the biological activity of theAHASL1 proteins. Thus, an isolated polynucleotide molecule encoding anAHASL1 protein having a sequence that differs from that of SEQ ID NOS: 1or 3, respectively, can be created by introducing one or more nucleotidesubstitutions, additions, or deletions into the corresponding nucleotidesequence disclosed herein, such that one or more amino acidsubstitutions, additions or deletions are introduced into the encodedprotein. Mutations can be introduced by standard techniques, such assite-directed mutagenesis and PCR-mediated mutagenesis. Such variantnucleotide sequences are also encompassed by the present invention.

For example, preferably, conservative amino acid substitutions may bemade at one or more predicted, preferably nonessential amino acidresidues. A “nonessential” amino acid residue is a residue that can bealtered from the wild-type sequence of an AHASL1 protein (e.g., thesequence of SEQ ID NO: 2) without altering the biological activity,whereas an “essential” amino acid residue is required for biologicalactivity. A “conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Such substitutions would not be made for conserved aminoacid residues, or for amino acid residues residing within a conservedmotif.

The proteins of the invention may be altered in various ways includingamino acid substitutions, deletions, truncations, and insertions.Methods for such manipulations are generally known in the art. Forexample, amino acid sequence variants of the AHASL1 proteins can beprepared by mutations in the DNA. Methods for mutagenesis and nucleotidesequence alterations are well known in the art. See, for example, Kunkel(1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987)Methods in Enzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walker andGaastra, eds. (1983) Techniques in Molecular Biology (MacMillanPublishing Company, New York) and the references cited therein. Guidanceas to appropriate amino acid substitutions that do not affect biologicalactivity of the protein of interest may be found in the model of Dayhoffet al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed.Res. Found., Washington, D.C.), herein incorporated by reference.Conservative substitutions, such as exchanging one amino acid withanother having similar properties, may be preferable.

Alternatively, variant AHASL1 nucleotide sequences can be made byintroducing mutations randomly along all or part of an AHASL1 codingsequence, such as by saturation mutagenesis, and the resultant mutantscan be screened for AHAS activity to identify mutants that retain AHASactivity, including herbicide-resistant AHAS activity. Followingmutagenesis, the encoded protein can be expressed recombinantly, and theactivity of the protein can be determined using standard assaytechniques.

Thus, the nucleotide sequences of the invention include the sequencesdisclosed herein as well as fragments and variants thereof. The AHASL1nucleotide sequences of the invention, and fragments and variantsthereof, can be used as probes and/or primers to identify and/or cloneAHASL homologues in other plants. Such probes can be used to detecttranscripts or genomic sequences encoding the same or identicalproteins.

In this manner, methods such as PCR, hybridization, and the like can beused to identify such sequences having substantial identity to thesequences of the invention. See, for example, Sambrook et al. (1989)Molecular Cloning: Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Plainview, N.Y.) and Innis, et al. (1990) PCRProtocols: A Guide to Methods and Applications (Academic Press, NY).AHASL nucleotide sequences isolated based on their sequence identity tothe AHASL1 nucleotide sequences set forth herein or to fragments andvariants thereof are encompassed by the present invention.

In a hybridization method, all or part of a known AHASL1 nucleotidesequence can be used to screen cDNA or genomic libraries. Methods forconstruction of such cDNA and genomic libraries are generally known inthe art and are disclosed in Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.). The so-called hybridization probes may be genomic DNAfragments, cDNA fragments, RNA fragments, or other oligonucleotides, andmay be labeled with a detectable group such as ³²P, or any otherdetectable marker, such as other radioisotopes, a fluorescent compound,an enzyme, or an enzyme co-factor. Probes for hybridization can be madeby labeling synthetic oligonucleotides based on the known AHASL1nucleotide sequence disclosed herein. Degenerate primers designed on thebasis of conserved nucleotides or amino acid residues in a known AHASL1nucleotide sequence or encoded amino acid sequence can additionally beused. The probe typically comprises a region of nucleotide sequence thathybridizes under stringent conditions to at least about 12, preferablyabout 25, more preferably about 50, 75, 100, 125, 150, 175, 200, 250,300, 350, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, or 1800consecutive nucleotides of an AHASL1 nucleotide sequence of theinvention or a fragment or variant thereof. Preparation of probes forhybridization is generally known in the art and is disclosed in Sambrooket al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., ColdSpring Harbor Laboratory Press, Plainview, N.Y.), herein incorporated byreference.

For example, the entire AHASL1 sequence disclosed herein, or one or moreportions thereof, may be used as a probe capable of specificallyhybridizing to corresponding AHASL1 sequences and messenger RNAs.Hybridization techniques include hybridization screening of plated DNAlibraries (either plaques or colonies; see, for example, Sambrook et al.(1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold SpringHarbor Laboratory Press, Plainview, N.Y.).

Hybridization of such sequences may be carried out under stringentconditions. By “stringent conditions” or “stringent hybridizationconditions” is intended conditions under which a probe will hybridize toits target sequence to a detectably greater degree than to othersequences (e.g., at least 2-fold over background). Stringent conditionsare sequence-dependent and will be different in different circumstances.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C. Optionally, wash buffersmay comprise about 0.1% to about 1% SDS. The duration of hybridizationis generally less than about 24 hours, usually about 4 to about 12hours.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284:T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization, and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with >90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence and its complement at a defined ionic strengthand pH. However, severely stringent conditions can utilize ahybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermalmelting point (T_(m)); moderately stringent conditions can utilize ahybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than thethermal melting point (T_(m)); low stringency conditions can utilize ahybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe thermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution), it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen (1993)Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2(Elsevier, N.Y.); and Ausubel et al., eds. (1995) Current Protocols inMolecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience,New York). See Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

It is recognized that the polynucleotide molecules and proteins of theinvention encompass polynucleotide molecules and proteins comprising anucleotide or an amino acid sequence that is sufficiently identical tothe nucleotide sequence of SEQ ID NOS: 1, 3, 4, and/or 6, or to theamino acid sequence of SEQ ID NOS: 2 and/or 5. The term “sufficientlyidentical” is used herein to refer to a first amino acid or nucleotidesequence that contains a sufficient or minimum number of identical orequivalent (e.g., with a similar side chain) amino acid residues ornucleotides to a second amino acid or nucleotide sequence such that thefirst and second amino acid or nucleotide sequences have a commonstructural domain and/or common functional activity. For example, aminoacid or nucleotide sequences that contain a common structural domainhaving at least about 45%, 55%, or 65% identity, preferably 75%identity, more preferably 85%, 95%, or 98% identity are defined hereinas sufficiently identical.

To determine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparisonpurposes. The percent identity between the two sequences is a functionof the number of identical positions shared by the sequences (i.e.,percent identity=number of identical positions/total number of positions(e.g., overlapping positions)×100). In one embodiment, the two sequencesare the same length. The percent identity between two sequences can bedetermined using techniques similar to those described below, with orwithout allowing gaps. In calculating percent identity, typically exactmatches are counted.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. A preferred, nonlimitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul (1990) Proc. Natl.Acad. Sci. USA 87:2264, modified as in Karlin and Altschul (1993) Proc.Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporatedinto the NBLAST and XBLAST programs of Altschul et al. (1990) J. Mol.Biol. 215:403. BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12, to obtain nucleotide sequenceshomologous to the polynucleotide molecules of the invention. BLASTprotein searches can be performed with the XBLAST program, score=50,wordlength=3, to obtain amino acid sequences homologous to proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.(1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-Blast can be usedto perform an iterated search that detects distant relationships betweenmolecules. See Altschul et al. (1997) supra. When utilizing BLAST,Gapped BLAST, and PSI-Blast programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used. Seehttp://www.ncbi.nlm.nih.gov. Another preferred, non-limiting example ofa mathematical algorithm utilized for the comparison of sequences is thealgorithm of Myers and Miller (1988) CABIOS 4:11-17. Such an algorithmis incorporated into the ALIGN program (version 2.0), which is part ofthe GCG sequence alignment software package. When utilizing the ALIGNprogram for comparing amino acid sequences, a PAM120 weight residuetable, a gap length penalty of 12, and a gap penalty of 4 can be used.Alignment may also be performed manually by inspection.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using the full-length sequences ofthe invention and using multiple alignment by mean of the algorithmClustal W (Nucleic Acid Research, 22(22):4673-4680, 1994) using theprogram AlignX included in the software package Vector NTI Suite Version7 (InforMax, Inc., Bethesda, Md., USA) using the default parameters; orany equivalent program thereof. By “equivalent program” is intended anysequence comparison program that, for any two sequences in question,generates an alignment having identical nucleotide or amino acid residuematches and an identical percent sequence identity when compared to thecorresponding alignment generated by AlignX in the software packageVector NTI Suite Version 7.

The AHASL1 nucleotide sequences of the invention include both thenaturally occurring sequences as well as mutant forms, particularlymutant forms that encode AHASL1 proteins comprising herbicide-resistantAHAS activity. Likewise, the proteins of the invention encompass bothnaturally occurring proteins as well as variations and modified formsthereof. Such variants will continue to possess the desired AHASactivity. Obviously, the mutations that will be made in the DNA encodingthe variant must not place the sequence out of reading frame andpreferably will not create complementary regions that could producesecondary mRNA structure. See, EP Patent Application Publication No.75,444.

The deletions, insertions, and substitutions of the protein sequencesencompassed herein are not expected to produce radical changes in thecharacteristics of the protein. However, when it is difficult to predictthe exact effect of the substitution, deletion, or insertion in advanceof doing so, one skilled in the art will appreciate that the effect willbe evaluated by routine screening assays. That is, the activity can beevaluated by AHAS activity assays. See, for example, Singh et al. (1988)Anal. Biochem. 171:173-179, herein incorporated by reference.

Variant nucleotide sequences and proteins also encompass sequences andproteins derived from a mutagenic and recombinogenic procedure such asDNA shuffling. With such a procedure, one or more different AHASL codingsequences can be manipulated to create a new AHASL protein possessingthe desired properties. In this manner, libraries of recombinantpolynucleotides are generated from a population of related sequencepolynucleotides comprising sequence regions that have substantialsequence identity and can be homologously recombined in vitro or invivo. For example, using this approach, sequence motifs encoding adomain of interest may be shuffled between the AHASL1 gene of theinvention and other known AHASL genes to obtain a new gene coding for aprotein with an improved property of interest, such as an increasedK_(m) in the case of an enzyme. Strategies for such DNA shuffling areknown in the art. See, for example, Stemmer (1994) Proc. Natl. Acad.Sci. USA 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri etal. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol.272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Pat.Nos. 5,605,793 and 5,837,458.

The nucleotide sequences of the invention can be used to isolatecorresponding sequences from other organisms, particularly other plants,more particularly other dicots. In this manner, methods such as PCR,hybridization, and the like can be used to identify such sequences basedon their sequence homology to the sequences set forth herein. Sequencesisolated based on their sequence identity to the entire AHASL1 sequencesset forth herein or to fragments thereof are encompassed by the presentinvention. Thus, isolated sequences that encode for an AHASL protein andwhich hybridize under stringent conditions to the sequence disclosedherein, or to fragments thereof, are encompassed by the presentinvention.

In a PCR approach, oligonucleotide primers can be designed for use inPCR reactions to amplify corresponding DNA sequences from cDNA orgenomic DNA extracted from any plant of interest. Methods for designingPCR primers and PCR cloning are generally known in the art and aredisclosed in Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods andApplications (Academic Press, New York); Innis and Gelfand, eds. (1995)PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds.(1999) PCR Methods Manual (Academic Press, New York). Known methods ofPCR include, but are not limited to, methods using paired primers,nested primers, single specific primers, degenerate primers,gene-specific primers, vector-specific primers, partially-mismatchedprimers, and the like.

The AHASL1 polynucleotide sequences of the invention are provided inexpression cassettes for expression in the plant of interest. Thecassette will include 5′ and 3′ regulatory sequences operably linked toan AHASL1 polynucleotide sequence of the invention. By “operably linked”is intended a functional linkage between a promoter and a secondsequence, wherein the promoter sequence initiates and mediatestranscription of the DNA sequence corresponding to the second sequence.Generally, operably linked means that the nucleic acid sequences beinglinked are contiguous and, where necessary to join two protein codingregions, contiguous and in the same reading frame. The cassette mayadditionally contain at least one additional gene to be cotransformedinto the organism. Alternatively, the additional gene(s) can be providedon multiple expression cassettes.

Such an expression cassette is provided with a plurality of restrictionsites for insertion of the AHASL1 polynucleotide sequence to be underthe transcriptional regulation of the regulatory regions. The expressioncassette may additionally contain selectable marker genes.

The expression cassette will include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region(i.e., a promoter), an AHASL1 polynucleotide sequence of the invention,and a transcriptional and translational termination region (i.e.,termination region) functional in plants. The promoter may be native oranalogous, or foreign or heterologous, to the plant host and/or to theAHASL1 polynucleotide sequence of the invention. Additionally, thepromoter may be the natural sequence or alternatively a syntheticsequence. Where the promoter is “foreign” or “heterologous” to the planthost, it is intended that the promoter is not found in the native plantinto which the promoter is introduced. Where the promoter is “foreign”or “heterologous” to the AHASL1 polynucleotide sequence of theinvention, it is intended that the promoter is not the native ornaturally occurring promoter for the operably linked AHASL1polynucleotide sequence of the invention. As used herein, a chimericgene comprises a coding sequence operably linked to a transcriptioninitiation region that is heterologous to the coding sequence.

While it may be preferable to express the AHASL1 polynucleotides of theinvention using heterologous promoters, the native promoter sequencesmay be used. Such constructs would change expression levels of theAHASL1 protein in the plant or plant cell. Thus, the phenotype of theplant or plant cell is altered.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked AHASL1 sequence ofinterest, may be native with the plant host, or may be derived fromanother source (i.e., foreign or heterologous to the promoter, theAHASL1 polynucleotide sequence of interest, the plant host, or anycombination thereof). Convenient termination regions are available fromthe Ti-plasmid of A. tumefaciens, such as the octopine synthase andnopaline synthase termination regions. See also Guerineau et al. (1991)Mol. Gen. Genet. 262:141444; Proudfoot (1991) Cell 64:671-674; Sanfaconet al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell2:1261-1272; Munroe et al. (1990) Gene 91:151-158; Ballas et al. (1989)Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic AcidRes. 15:9627-9639.

Where appropriate, the gene(s) may be optimized for increased expressionin the transformed plant. That is, the genes can be synthesized usingplant-preferred codons for improved expression. See, for example,Campbell and Gown (1990) Plant Physiol. 92:1-11 for a discussion ofhost-preferred codon usage. Methods are available in the art forsynthesizing plant-preferred genes. See, for example, U.S. Pat. Nos.5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic Acids Res.17:477-498, herein incorporated by reference.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well-characterized sequencesthat may be deleterious to gene expression. The G-C content of thesequence may be adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Whenpossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures.

Nucleotide sequences for enhancing gene expression can also be used inthe plant expression vectors. These include the introns of the maizeAdhI, intron1 gene (Callis et al. Genes and Development 1:1183-1200,1987), and leader sequences, (W-sequence) from the Tobacco Mosaic virus(TMV), Maize Chlorotic Mottle Virus and Alfalfa Mosaic Virus (Gallie etal. Nucleic Acid Res. 15:8693-8711, 1987 and Skuzeski et al. Plant Mol.Biol. 15:65-79, 1990). The first intron from the shrunken-1 locus ofmaize, has been shown to increase expression of genes in chimeric geneconstructs. U.S. Pat. Nos. 5,424,412 and 5,593,874 disclose the use ofspecific introns in gene expression constructs, and Gallie et al. (PlantPhysiol. 106:929-939, 1994) also have shown that introns are useful forregulating gene expression on a tissue specific basis. To furtherenhance or to optimize AHAS small subunit gene expression, the plantexpression vectors of the invention may also contain DNA sequencescontaining matrix attachment regions (MARS). Plant cells transformedwith such modified expression systems, then, may exhibit overexpressionor constitutive expression of a nucleotide sequence of the invention.

The expression cassettes may additionally contain 5′ leader sequences inthe expression cassette construct. Such leader sequences can act toenhance translation. Translation leaders are known in the art andinclude: picornavirus leaders, for example, EMCV leader(Encephalomyocarditis 5′ noncoding region) (Elroy-Stein et al. (1989)Proc. Natl. Acad. Sci. USA 86:6126-6130); potyvirus leaders, forexample, TEV leader (Tobacco Etch Virus) (Gallie et al. (1995) Gene165(2):233-238), MDMV leader (Maize Dwarf Mosaic Virus) (Virology154:9-20), and human immunoglobulin heavy-chain binding protein (BiP)(Macejak et al. (1991) Nature 353:90-94); untranslated leader from thecoat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al.(1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie etal. (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp.237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel et al.(1991) Virology 81:382-385). See also, Della-Cioppa et al. (1987) PlantPhysiol. 84:965-968. Other methods known to enhance translation can alsobe utilized, for example, introns, and the like.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

A number of promoters can be used in the practice of the invention. Thepromoters can be selected based on the desired outcome. The nucleicacids can be combined with constitutive, tissue-preferred, or otherpromoters for expression in plants.

Such constitutive promoters include, for example, the core promoter ofthe Rsyn7 promoter and other constitutive promoters disclosed in WO99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odellet al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990)Plant Cell 2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol.Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol.18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588);MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat.No. 5,659,026), and the like. Other constitutive promoters include, forexample, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597;5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.

Tissue-preferred promoters can be utilized to target enhanced AHASL1expression within a particular plant tissue. Such tissue-preferredpromoters include, but are not limited to, leaf-preferred promoters,root-preferred promoters, seed-preferred promoters, and stem-preferredpromoters. Tissue-preferred promoters include Yamamoto et al. (1997)Plant J. 12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol.38(7):792-803; Hansen et al. (1997) Mol. Gen Genet. 254(3):337-343;Russell et al. (1997) Transgenic Res. 6(2):157-168; Rinehart et al.(1996) Plant Physiol. 112(3):1331-1341; Van Camp et al. (1996) PlantPhysiol. 112(2):525-535; Canevascini et al. (1996) Plant Physiol.112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol.35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196; Orozcoet al. (1993) Plant Mol Biol. 23(6):1129-1138; Matsuoka et al. (1993)Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-Garcia et al.(1993) Plant J. 4(3):495-505. Such promoters can be modified, ifnecessary, for weak expression.

In one embodiment, the nucleic acids of interest are targeted to thechloroplast for expression. In this manner, where the nucleic acid ofinterest is not directly inserted into the chloroplast, the expressioncassette will additionally contain a chloroplast-targeting sequencecomprising a nucleotide sequence that encodes a chloroplast transitpeptide to direct the gene product of interest to the chloroplasts.

Such transit peptides are known in the art. With respect tochloroplast-targeting sequences, “operably linked” means that thenucleic acid sequence encoding a transit peptide (i.e., thechloroplast-targeting sequence) is linked to the AHASL1 polynucleotideof the invention such that the two sequences are contiguous and in thesame reading frame. See, for example, Von Heijne et al. (1991) PlantMol. Biol. Rep. 9:104-126; Clark et al. (1989) J. Biol. Chem.264:17544-17550; Della-Cioppa et al. (1987) Plant Physiol. 84:965-968;Romer et al. (1993) Biochem. Biophys. Res. Commun. 196:1414-1421; andShah et al. (1986) Science 233:478-481. While the AHASL1 proteins of theinvention include a native chloroplast transit peptide, any chloroplasttransit peptide known in art can be fused to the amino acid sequence ofa mature AHASL1 protein of the invention by operably linking achloroplast-targeting sequence to the 5′-end of a nucleotide sequenceencoding a mature AHASL1 protein of the invention.

Chloroplast targeting sequences are known in the art and include thechloroplast small subunit of ribulose-1,5-bisphosphate carboxylase(Rubisco) (de Castro Silva Fitho et al. (1996) Plant Mol. Biol.30:769-780; Schnell et al. (1991) J. Biol. Chem. 266(5):3335-3342);5-(enolpyruvyl)shikimate-3-phosphate synthase (EPSPS) (Archer et al.(1990) J. Bioenerg. Biomemb. 22(6):789-810); tryptophan synthase (Zhaoet al. (1995) J. Biol. Chem. 270(11):6081-6087); plastocyanin (Lawrenceet al. (1997) J. Biol. Chem. 272(33):20357-20363); chorismate synthase(Schmidt et al. (1993) J. Biol. Chem. 268(36):27447-27457); and thelight harvesting chlorophyll a/b binding protein (LHBP) (Lamppa et al.(1988) J. Biol. Chem. 263:14996-14999). See also Von Heijne et al.(1991) Plant Mol. Biol. Rep. 9:104-126; Clark et al. (1989) J. Biol.Chem. 264:17544-17550; Della-Cioppa et al. (1987) Plant Physiol.84:965-968; Romer et al. (1993) Biochem. Biophys. Res. Commun.196:1414-1421; and Shah et al. (1986) Science 233:478-481.

Methods for transformation of chloroplasts are known in the art. See,for example, Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530;Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab andMaliga (1993) EMBO J. 12:601-606. The method relies on particle gundelivery of DNA containing a selectable marker and targeting of the DNAto the plastid genome through homologous recombination. Additionally,plastid transformation can be accomplished by transactivation of asilent plastid-borne transgene by tissue-preferred expression of anuclear-encoded and plastid-directed RNA polymerase. Such a system hasbeen reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA91:7301-7305.

The nucleic acids of interest to be targeted to the chloroplast may beoptimized for expression in the chloroplast to account for differencesin codon usage between the plant nucleus and this organelle. In thismanner, the nucleic acids of interest may be synthesized usingchloroplast-preferred codons. See, for example, U.S. Pat. No. 5,380,831,herein incorporated by reference.

As disclosed herein, the AHASL1 nucleotide sequences of the inventionfind use in enhancing the herbicide tolerance of plants that comprise intheir genomes a gene encoding a herbicide-tolerant AHASL1 protein. Sucha gene may be an endogenous gene or a transgene. Additionally, incertain embodiments, the nucleic acid sequences of the present inventioncan be stacked with any combination of polynucleotide sequences ofinterest in order to create plants with a desired phenotype. Forexample, the polynucleotides of the present invention may be stackedwith any other polynucleotides encoding polypeptides having pesticidaland/or insecticidal activity, such as, for example, the Bacillusthuringiensis toxin proteins (described in U.S. Pat. Nos. 5,366,892;5,747,450; 5,737,514; 5,723,756; 5,593,881; and Geiser et al. (1986)Gene 48:109). The combinations generated can also include multiplecopies of any one of the polynucleotides of interest.

It is recognized that with these nucleotide sequences, antisenseconstructions, complementary to at least a portion of the messenger RNA(mRNA) for the AHASL1 polynucleotide sequences can be constructed.Antisense nucleotides are constructed to hybridize with thecorresponding mRNA. Modifications of the antisense sequences may be madeas long as the sequences hybridize to and interfere with expression ofthe corresponding mRNA. In this manner, antisense constructions having70%, preferably 80%, more preferably 85% sequence identity to thecorresponding antisensed sequences may be used. Furthermore, portions ofthe antisense nucleotides may be used to disrupt the expression of thetarget gene. Generally, sequences of at least 50 nucleotides, 100nucleotides, 200 nucleotides, or greater may be used.

The nucleotide sequences of the present invention may also be used inthe sense orientation to suppress the expression of endogenous genes inplants. Methods for suppressing gene expression in plants usingnucleotide sequences in the sense orientation are known in the art. Themethods generally involve transforming plants with a DNA constructcomprising a promoter that drives expression in a plant operably linkedto at least a portion of a nucleotide sequence that corresponds to thetranscript of the endogenous gene. Typically, such a nucleotide sequencehas substantial sequence identity to the sequence of the transcript ofthe endogenous gene, preferably greater than about 65% sequenceidentity, more preferably greater than about 85% sequence identity, mostpreferably greater than about 95% sequence identity. See, U.S. Pat. Nos.5,283,184 and 5,034,323; herein incorporated by reference.

While the herbicide-resistant AHASL1 polynucleotides of the inventionfind use as selectable marker genes for plant transformation, theexpression cassettes of the invention can include another selectablemarker gene for the selection of transformed cells. Selectable markergenes, including those of the present invention, are utilized for theselection of transformed cells or tissues. Marker genes include, but arenot limited to, genes encoding antibiotic resistance, such as thoseencoding neomycin phosphotransferase II (NEO) and hygromycinphosphotransferase (HPT), as well as genes conferring resistance toherbicidal compounds, such as glufosinate ammonium, bromoxynil,imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). See generally,Yarranton (1992) Curr. Opin. Biotech. 3:506-511; Christopherson et al.(1992) Proc. Natl. Acad. Sci. USA 89:6314-6318; Yao et al. (1992) Cell71:63-72; Reznikoff (1992) Mol. Microbiol. 6:2419-2422; Barkley et al.(1980) in The Operon, pp. 177-220; Hu et al. (1987) Cell 48:555-566;Brown et al. (1987) Cell 49:603-612; Figge et al. (1988) Cell52:713-722; Deuschle et al. (1989) Proc. Natl. Acad. Aci. USA86:5400-5404; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA86:2549-2553; Deuschle et al. (1990) Science 248:480-483; Gossen (1993)Ph.D. Thesis, University of Heidelberg; Reines et al. (1993) Proc. Natl.Acad. Sci. USA 90:1917-1921; Labow et al. (1990) Mol. Cell. Biol.10:3343-3356; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA89:3952-3956; Baim et al. (1991) Proc. Natl. Acad. Sci. USA88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res. 19:4647-4653;Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10:143-162; Degenkolbet al. (1991) Antimicrob. Agents Chemother. 35:1591-1595; Kleinschnidtet al. (1988) Biochemistry 27:1094-1104; Bonin (1993) Ph.D. Thesis,University of Heidelberg; Gossen et al. (1992) Proc. Natl. Acad. Sci.USA 89:5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother.36:913-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology,Vol. 78 (Springer-Verlag, Berlin); Gill et al. (1988) Nature334:721-724. Such disclosures are herein incorporated by reference.

The above list of selectable marker genes is not meant to be limiting.Any selectable marker gene can be used in the present invention.

The isolated polynucleotide molecules comprising nucleotide sequencethat encode the AHASL1 proteins of the invention can be used in vectorsto transform plants so, that the plants created have enhanced resistantto herbicides, particularly imidazolinone herbicides. The isolatedAHASL1 polynucleotide molecules of the invention can be used in vectorsalone or in combination with a nucleotide sequence encoding the smallsubunit of the AHAS (AHASS) enzyme in conferring herbicide resistance inplants. See, U.S. Pat. No. 6,348,643; which is herein incorporated byreference.

The invention also relates to a plant expression vector comprising apromoter that drives expression in a plant operably linked to anisolated polynucleotide molecule of the invention. The isolatedpolynucleotide molecule comprises a nucleotide sequence encoding anAHASL1 protein, particularly an AHASL1 protein comprising an aminosequence that is set forth in SEQ ID NO: 2, or a functional fragment andvariant thereof. The plant expression vector of the invention does notdepend on a particular promoter, only that such a promoter is capable ofdriving gene expression in a plant cell. Preferred promoters includeconstitutive promoters and tissue-preferred promoters.

The transformation vectors of the invention can be used to produceplants transformed with a gene of interest. The transformation vectorwill comprise a selectable marker gene of the invention and a gene ofinterest to be introduced and typically expressed in the transformedplant. Such a selectable marker gene comprises a herbicide-resistantAHASL1 polynucleotide of the invention operably linked to a promoterthat drives expression in a host cell. For use in plants and plantcells, the transformation vector comprises a selectable marker genecomprising a herbicide-resistant AHASL1 polynucleotide of the inventionoperably linked to a promoter that drives expression in a plant cell.

The genes of interest of the invention vary depending on the desiredoutcome. For example, various changes in phenotype can be of interestincluding modifying the fatty acid composition in a plant, altering theamino acid content of a plant, altering a plant's insect and/or pathogendefense mechanisms, and the like. These results can be achieved byproviding expression of heterologous products or increased expression ofendogenous products in plants. Alternatively, the results can beachieved by providing for a reduction of expression of one or moreendogenous products, particularly enzymes or cofactors in the plant.These changes result in a change in phenotype of the transformed plant.

In one embodiment of the invention, the genes of interest include insectresistance genes such as, for example, Bacillus thuringiensis toxinprotein genes (U.S. Pat. Nos. 5,366,892; 5,747,450; 5,736,514;5,723,756; 5,593,881; and Geiser et al. (1986) Gene 48:109).

The AHASL1 proteins or polypeptides of the invention can be purifiedfrom, for example, rice plants and can be used in compositions. Also, anisolated polynucleotide molecule encoding an AHASL1 protein of theinvention can be used to express an AHASL1 protein of the invention in amicrobe such as E. coli or a yeast. The expressed AHASL1 protein can bepurified from extracts of E. coli or yeast by any method known to thoseor ordinary skill in the art.

The invention also relates to a method for creating a transgenic plantthat is resistant to herbicides, comprising transforming a plant with aplant expression vector comprising a promoter that drives expression ina plant operably linked to an isolated polynucleotide molecule of theinvention. The isolated polynucleotide molecule comprises a nucleotidesequence encoding an AHASL1 protein of the invention, particularly anAHASL1 protein comprising: an amino sequence that is set forth in SEQ IDNO: 2, an amino acid sequence encoded by SEQ ID NO: 1 or 3, or afunctional fragment and variant of said amino acid sequences.

The invention also relates to the non-transgenic rice plants, transgenicplants produced by the methods of the invention, and progeny and otherdescendants of such non-transgenic and transgenic plants, which plantsexhibit enhanced or increased resistance to herbicides that interferewith the AHAS enzyme, particularly imidazolinone and sulfonylureaherbicides.

The AHASL1 polynucleotides of the invention, particularly those encodingherbicide-resistant AHASL1 proteins, find use in methods for enhancingthe resistance of herbicide-tolerant plants. In one embodiment of theinvention, the herbicide-tolerant plants comprise a herbicide-tolerantor herbicide resistant AHASL1 protein. The herbicide-tolerant plantsinclude both plants transformed with a herbicide-tolerant AHASL1nucleotide sequences and plants that comprise in their genomes anendogenous gene that encodes a herbicide-tolerant AHASL1 protein.Nucleotide sequences encoding herbicide-tolerant AHASL1 proteins andherbicide-tolerant plants comprising an endogenous gene that encodes aherbicide-tolerant AHASL1 protein include the polynucleotides and plantsof the present invention and those that are known in the art. See, forexample, U.S. Pat. Nos. 5,013,659, 5,731,180, 5,767,361, 5,545,822,5,736,629, 5,773,703, 5,773,704, 5,952,553 and 6,274,796; all of whichare herein incorporated by reference. Such methods for enhancing theresistance of herbicide-tolerant plants comprise transforming aherbicide-tolerant plant with at least one polynucleotide constructioncomprising a promoter that drives expression in a plant cell that isoperably linked to a herbicide resistant AHASL1 polynucleotide of theinvention, particularly the polynucleotide encoding aherbicide-resistant AHASL1 protein set forth in SEQ ID NO: 1 or 3polynucleotides encoding the amino acid sequence set forth in SEQ ID NO:2, and fragments and variants said polynucleotides that encodepolypeptides comprising herbicide-resistant AHAS activity.

Numerous plant transformation vectors and methods for transformingplants are available. See, for example, An, G. et al. (1986) PlantPysiol., 81:301-305; Fry, J., et al. (1987) Plant Cell Rep. 6:321-325;Block, M. (1988) Theor. Appl. Genet.76:767-774; Hinchee, et al. (1990)Stadler. Genet. Symp. 203212.203-212; Cousins, et al. (1991) Aust. J.Plant Physiol. 18:481-494; Chee, P. P. and Slightom, J. L. (1992)Gene.118:255-260; Christou, et al. (1992) Trends. Biotechnol.10:239-246; D'Halluin, et al. (1992) Bio/Technol. 10:309-314; Dhir, etal. (1992) Plant Physiol. 99:81-88; Casas et al. (1993) Proc. Nat. AcadSci. USA 90:11212-11216; Christou, P. (1993) In Vitro Cell. Dev.Biol.-Plant; 29P:119-124; Davies, et al. (1993) Plant Cell Rep.12:180-183; Dong, J. A. and Mchughen, A. (1993) Plant Sci. 91:139-148;Franklin, C. I. and Trieu, T. N. (1993) Plant. Physiol. 102:167;Golovkin, et al. (1993) Plant Sci. 90:41-52; Guo Chin Sci. Bull.38:2072-2078; Asano, et al. (1994) Plant Cell Rep. 13; Ayeres N. M. andPark, W. D. (1994) Crit. Rev. Plant. Sci. 13:219-239; Barcelo, et al.(1994) Plant. J. 5:583-592; Becker, et al. (1994) Plant. J. 5:299-307;Borkowska et al. (1994) Acta. Physiol Plant. 16:225-230; Christou, P.(1994) Agro. Food. Ind. Hi Tech. 5: 17-27; Eapen et al. (1994) PlantCell Rep. 13:582-586; Hartman, et al. (1994) Bio-Technology 12: 919923;Ritala, et al. (1994) Plant. Mol. Biol. 24:317-325; and Wan, Y. C. andLemaux, P. G. (1994) Plant Physiol. 104:3748.

The methods of the invention involve introducing a polynucleotideconstruct into a plant. By “introducing” is intended presenting to theplant the polynucleotide construct in such a manner that the constructgains access to the interior of a cell of the plant. The methods of theinvention do not depend on a particular method for introducing apolynucleotide construct to a plant, only that the polynucleotideconstruct gains access to the interior of at least one cell of theplant. Methods for introducing polynucleotide constructs into plants areknown in the art including, but not limited to, stable transformationmethods, transient transformation methods, and virus-mediated methods.

By “stable transformation” is intended that the polynucleotide constructintroduced into a plant integrates into the genome of the plant and iscapable of being inherited by progeny thereof. By “transienttransformation” is intended that a polynucleotide construct introducedinto a plant does not integrate into the genome of the plant.

For the transformation of plants and plant cells, the nucleotidesequences of the invention are inserted using standard techniques intoany vector known in the art that is suitable for expression of thenucleotide sequences in a plant or plant cell. The selection of thevector depends on the preferred transformation technique and the targetplant species to be transformed. In an embodiment of the invention, anAHASL1 nucleotide sequence is operably linked to a plant promoter thatis known for high-level expression in a plant cell, and this constructis then introduced into a plant that that is susceptible to animidazolinone herbicide and a transformed plant it regenerated. Thetransformed plant is tolerant to exposure to a level of an imidazolinoneherbicide that would kill or significantly injure an untransformedplant. This method can be applied to any plant species; however, it ismost beneficial when applied to crop plants, particularly crop plantsthat are typically grown in the presence of at least one herbicide,particularly an imidazolinone herbicide.

Methodologies for constructing plant expression cassettes andintroducing foreign nucleic acids into plants are generally known in theart and have been previously described. For example, foreign DNA can beintroduced into plants, using tumor-inducing (Ti) plasmid vectors. Othermethods utilized for foreign DNA delivery involve the use of PEGmediated protoplast transformation, electroporation, microinjectionwhiskers, and biolistics or microprojectile bombardment for direct DNAuptake. Such methods are known in the art. (U.S. Pat. No. 5,405,765 toVasil et al.; Bilang et al. (1991) Gene 100: 247-250; Scheid et al.,(1991) Mol. Gen. Genet., 228: 104-112; Guerche et al., (1987) PlantScience 52: 111-116; Neuhause et al., (1987) Theor. Appl. Genet. 75:30-36; Klein et al., (1987) Nature 327: 70-73; Howell et al., (1980)Science 208:1265; Horsch et al., (1985) Science 227: 1229-1231; DeBlocket al., (1989) Plant Physiology 91: 694-701; Methods for Plant MolecularBiology (Weissbach and Weissbach, eds.) Academic Press, Inc. (1988) andMethods in Plant Molecular Biology (Schuler and Zielinski, eds.)Academic Press, Inc. (1989). The method of transformation depends uponthe plant cell to be transformed, stability of vectors used, expressionlevel of gene products and other parameters.

Other suitable methods of introducing nucleotide sequences into plantcells and subsequent insertion into the plant genome includemicroinjection as Crossway et al. (1986) Biotechniques 4:320-334,electroporation as described by Riggs et al. (1986) Proc. Natl. Acad.Sci. USA 83:5602-5606, Agrobacterium-mediated transformation asdescribed by Townsend et al., U.S. Pat. No. 5,563,055, Zhao et al., U.S.Pat. No. 5,981,840, direct gene transfer as described by Paszkowski etal. (1984) EMBO J 3:2717-2722, and ballistic particle acceleration asdescribed in, for example, Sanford et al., U.S. Pat. No. 4,945,050;Tomes et al., U.S. Pat. No. 5,879,918; Tomes et al., U.S. Pat. No.5,886,244; Bidney et al., U.S. Pat. No. 5,932,782; Tomes et al. (1995)“Direct DNA Transfer into Intact Plant Cells via MicroprojectileBombardment,” in Plant Cell, Tissue, and Organ Culture: FundamentalMethods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); McCabe etal. (1988) Biotechnology 6:923-926); and Lec1 transformation (WO00/28058). Also see, Weissinger et al. (1988) Ann. Rev. Genet.22:421-477; Sanford et al. (1987) Particulate Science and Technology5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87:671-674(soybean); McCabe et al. (1988) Bio/Technology 6:923-926 (soybean);Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P:175-182(soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (soybean);Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. (1988)Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988)Biotechnology 6:559-563 (maize); Tomes, U.S. Pat. No. 5,240,855; Buisinget al., U.S. Pat. Nos. 5,322,783 and 5,324,646; Tomes et al. (1995)“Direct DNA Transfer into Intact Plant Cells via MicroprojectileBombardment,” in Plant Cell, Tissue, and Organ Culture: FundamentalMethods, ed. Gamborg (Springer-Verlag, Berlin) (maize); Klein et al.(1988) Plant Physiol. 91:440-444 (maize); Fromm et al. (1990)Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al. (1984)Nature (London) 311:763-764; Bowen et al., U.S. Pat. No. 5,736,369(cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA84:5345-5349 (Liliaceae); De Wet et al. (1985) in The ExperimentalManipulation of Ovule Tissues, ed. Chapman et al. (Longman, N.Y.), pp.197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566(whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413(rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize viaAgrobacterium tumefaciens); all of which are herein incorporated byreference.

The polynucleotides of the invention may be introduced into plants bycontacting plants with a virus or viral nucleic acids. Generally, suchmethods involve incorporating a polynucleotide construct of theinvention within a viral DNA or RNA molecule. It is recognized that thean AHASL1 protein of the invention may be initially synthesized as partof a viral polyprotein, which later may be processed by proteolysis invivo or in vitro to produce the desired recombinant protein. Further, itis recognized that promoters of the invention also encompass promotersutilized for transcription by viral RNA polymerases. Methods forintroducing polynucleotide constructs into plants and expressing aprotein encoded therein, involving viral DNA or RNA molecules, are knownin the art. See, for example, U.S. Pat. Nos. 5,889,191, 5,889,190,5,866,785, 5,589,367 and 5,316,931; herein incorporated by reference.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, the present invention provides transformed seed (alsoreferred to as “transgenic seed”) having a polynucleotide construct ofthe invention, for example, an expression cassette of the invention,stably incorporated into their genome.

The present invention may be used for transformation of any plantspecies, including, but not limited to, monocots and dicots. Examples ofplant species of interest include, but are not limited to, corn or maize(Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea),particularly those Brassica species useful as sources of seed oil,alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale),sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet(Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet(Setaria italica), finger millet (Eleusine coracana), sunflower(Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticumaestivum, T. Turgidum ssp. durum), soybean (Glycine max), tobacco(Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachishypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweetpotato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffeaspp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrustrees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis),banana (Musa spp.), avocado (Persea americana), fig (Ficus casica),guava (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), papaya (Carica papaya), cashew (Anzacardium occidentale),macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugarbeets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley,vegetables, ornamentals, and conifers. Preferably, plants of the presentinvention are crop plants (for example, sunflower, Brassica sp., cotton,sugar, beet, soybean, peanut, alfalfa, safflower, tobacco, corn, rice,wheat, rye, barley triticale, sorghum, millet, etc.).

The herbicide resistant plants of the invention find use in methods forcontrolling weeds. Thus, the present invention further provides a methodfor controlling weeds in the vicinity of a herbicide-resistant plant ofthe invention. The method comprises applying an effective amount of aherbicide to the weeds and to the herbicide-resistant plant, wherein theplant has increased resistance to at least one herbicide, particularlyan imidazolinone or sulfonylurea herbicide, when compared to a wild-typeplant. In such a method for controlling weeds, the herbicide-resistantplants of the invention are preferably crop plants, including, but notlimited to, rice, sunflower, alfalfa, Brassica sp., soybean, cotton,safflower, peanut, tobacco, tomato, potato, wheat, maize, sorghum,barley, rye, millet, and sorghum.

By providing plants having increased resistance to herbicides,particularly imidazolinone and sulfonylurea herbicides, a wide varietyof formulations can be employed for protecting plants from weeds, so asto enhance plant growth and reduce competition for nutrients. Aherbicide can be used by itself for pre-emergence, post-emergence,pre-planting and at planting control of weeds in areas surrounding theplants described herein or an imidazolinone herbicide formulation can beused that contains other additives. The herbicide can also be used as aseed treatment. Additives found in an imidazolinone or sulfonylureaherbicide formulation include other herbicides, detergents, adjuvants,spreading agents, sticking agents, stabilizing agents, or the like. Theherbicide formulation can be a wet or dry preparation and can include,but is not limited to, flowable powders, emulsifiable concentrates andliquid concentrates. The herbicide and herbicide formulations can beapplied in accordance with conventional methods, for example, byspraying, irrigation, dusting, or the like.

The present invention provides non-transgenic and transgenic seeds withincreased tolerance to at least one herbicide, particularly anAHAS-inhibiting herbicide, more particularly an imidazolinone herbicide.Such seeds include, for example, non-transgenic rice seeds comprisingthe herbicide-tolerance characteristics of the plant with NCIMBAccession Number NCIMB 41262, and transgenic seeds comprising an IMInucleic acid molecule of the invention that encodes an IMI protein.

The present invention provides methods for producing aherbicide-resistant plant, particularly a herbicide-resistant riceplant, through conventional plant breeding involving sexualreproduction. The methods comprise crossing a first plant that isresistant to a herbicide to a second plant that is not resistant to theherbicide. The first plant can be any of the herbicide resistant plantsof the present invention including, for example, transgenic plantscomprising at least one of the polynucleotides of the present inventionthat encode a herbicide resistant IMI protein and non-transgenic riceplants that comprise the herbicide-tolerance characteristics of the riceplant with NCIMB Accession Number NCIMB 41262. The second plant can beany plant that is capable of producing viable progeny plants (i.e.,seeds) when crossed with the first plant. Typically, but notnecessarily, the first and second plants are of the same species. Themethods of the invention can further involve one or more generations ofbackcrossing the progeny plants of the first cross to a plant of thesame line or genotype as either the first or second plant.Alternatively, the progeny of the first cross or any subsequent crosscan be crossed to a third plant that is of a different line or genotypethan either the first or second plant. The methods of the invention canadditionally involve selecting plants that comprise the herbicidetolerance characteristics of the first plant.

The present invention further provides methods for increasing theherbicide-resistance of a plant, particularly a herbicide-resistant riceplant, through conventional plant breeding involving sexualreproduction. The methods comprise crossing a first plant that isresistant to a herbicide to a second plant that may or may not beresistant to the herbicide or may be resistant to different herbicide orherbicides than the first plant. The first plant can be any of theherbicide resistant plants of the present invention including, forexample, transgenic plants comprising at least one of the IMI nucleicacids of the present invention that encode IMI protein andnon-transgenic rice plants that comprise the herbicide-tolerancecharacteristics of the rice plant with NCIMB Accession Number NCIMB41262. The second plant can be any plant that is capable of producingviable progeny plants (i.e., seeds) when crossed with the first plant.Typically, but not necessarily, the first and second plants are of thesame species. The progeny plants produced by this method of the presentinvention have increased resistance to a herbicide when compared toeither the first or second plant or both. When the first and secondplants are resistant to different herbicides, the progeny plants willhave the combined herbicide tolerance characteristics of the first andsecond plants. The methods of the invention can further involve one ormore generations of backcrossing the progeny plants of the first crossto a plant of the same line or genotype as either the first or secondplant. Alternatively, the progeny of the first cross or any subsequentcross can be crossed to a third plant that is of a different line orgenotype than either the first or second plant. The methods of theinvention can additionally involve selecting plants that comprise theherbicide tolerance characteristics of the first plant, the secondplant, or both the first and the second plant.

The plants of the present invention can be transgenic or non-transgenic.An example of a non-transgenic rice plant having increased resistance toimidazolinone is the rice plant (IMINTA 16) having NCIMB AccessionNumber NCIMB 41262; or mutant, recombinant, or a genetically engineeredderivative of the plant having NCIMB Accession Number NCIMB 41262; or ofany progeny of the plant having NCIMB Accession Number NCIMB 41262; or aplant that is a progeny of any of these plants; or a plant thatcomprises the herbicide tolerance characteristics of the plant havingNCIMB Accession Number NCIMB 41262.

The present invention also provides plants, plant organs, plant tissues,plant cells, seeds, and non-human host cells that are transformed withthe at least one polynucleotide molecule, expression cassette, ortransformation vector of the invention. Such transformed plants, plantorgans, plant tissues, plant cells, seeds, and non-human host cells haveenhanced tolerance or resistance to at least one herbicide, at levels ofthe herbicide that kill or inhibit the growth of an untransformed plant,plant tissue, plant cell, or non-human host cell, respectively.Preferably, the transformed plants, plant tissues, plant cells, andseeds of the invention are Arabidopsis thaliana and crop plants.

The present invention provides methods that involve the use of at leastone AHAS-inhibiting herbicide selected from the group consisting ofimidazolinone herbicides, sulfonylurea herbicides, triazolopyrimidineherbicides, pyrimidinyloxybenzoate herbicides,sulfonylamino-carbonyltriazolinone herbicides, and mixtures thereof. Inthese methods, the AHAS-inhibiting herbicide can be applied by anymethod known in the art including, but not limited to, seed treatment,soil treatment, and foliar treatment.

Prior to application, the AHAS-inhibiting herbicide can be convertedinto the customary formulations, for example solutions, emulsions,suspensions, dusts, powders, pastes and granules. The use form dependson the particular intended purpose; in each case, it should ensure afine and even distribution of the compound according to the invention.

The formulations are prepared in a known manner (see e.g. for reviewU.S. Pat. No. 3,060,084, EP-A 707 445 (for liquid concentrates),Browning, “Agglomeration”, Chemical Engineering, Dec. 4, 1967, 147-48,Perry's Chemical Engineer's Handbook, 4th Ed., McGraw-Hill, New York,1963, pages 8-57 and et seq. WO 91/13546, U.S. Pat. No. 4,172,714, U.S.Pat. No. 4,144,050, U.S. Pat. No. 3,920,442, U.S. Pat. No. 5,180,587,U.S. Pat. No. 5,232,701, U.S. Pat. No. 5,208,030, GB 2,095,558, U.S.Pat. No. 3,299,566, Klingman, Weed Control as a Science, John Wiley andSons, Inc., New York, 1961, Hance et al., Weed Control Handbook, 8thEd., Blackwell Scientific Publications, Oxford, 1989 and Mollet, H.,Grubemann, A., Formulation technology, Wiley VCH Verlag GmbH, Weinheim(Germany), 2001, 2. D. A. Knowles, Chemistry and Technology ofAgrochemical Formulations, Kluwer Academic Publishers, Dordrecht, 1998(ISBN 0-7514-0443-8), for example by extending the active compound withauxiliaries suitable for the formulation of agrochemicals, such assolvents and/or carriers, if desired emulsifiers, surfactants anddispersants, preservatives, antifoaming agents, anti-freezing agents,for seed treatment formulation also optionally colorants and/or bindersand/or gelling agents.

Examples of suitable solvents are water, aromatic solvents (for exampleSolvesso products, xylene), paraffins (for example mineral oilfractions), alcohols (for example methanol, butanol, pentanol, benzylalcohol), ketones (for example cyclohexanone, gamma-butyrolactone),pyrrolidones (NMP, NOP), acetates (glycol diacetate), glycols, fattyacid dimethylamides, fatty acids and fatty acid esters. In principle,solvent mixtures may also be used.

Examples of suitable carriers are ground natural minerals (for examplekaolins, clays, talc, chalk) and ground synthetic minerals (for examplehighly disperse silica, silicates).

Suitable emulsifiers are nonionic and anionic emulsifiers (for examplepolyoxyethylene fatty alcohol ethers, alkylsulfonates andarylsulfonates).

Examples of dispersants are lignin-sulfite waste liquors andmethylcellulose.

Suitable surfactants used are alkali metal, alkaline earth metal andammonium salts of lignosulfonic acid, naphthalenesulfonic acid,phenolsulfonic acid, dibutylnaphthalenesulfonic acid,alkylarylsulfonates, alkyl sulfates, alkylsulfonates, fatty alcoholsulfates, fatty acids and sulfated fatty alcohol glycol ethers,furthermore condensates of sulfonated naphthalene and naphthalenederivatives with formaldehyde, condensates of naphthalene or ofnaphthalenesulfonic acid with phenol and formaldehyde, polyoxyethyleneoctylphenol ether, ethoxylated isooctylphenol, octylphenol, nonylphenol,alkylphenol polyglycol ethers, tributylphenyl polyglycol ether,tristearylphenyl polyglycol ether, alkylaryl polyether alcohols, alcoholand fatty alcohol ethylene oxide condensates, ethoxylated castor oil,polyoxyethylene alkyl ethers, ethoxylated polyoxypropylene, laurylalcohol polyglycol ether acetal, sorbitol esters, lignosulfite wasteliquors and methylcellulose.

Substances which are suitable for the preparation of directly sprayablesolutions, emulsions, pastes or oil dispersions are mineral oilfractions of medium to high boiling point, such as kerosene or dieseloil, furthermore coal tar oils and oils of vegetable or animal origin,aliphatic, cyclic and aromatic hydrocarbons, for example toluene,xylene, paraffin, tetrahydronaphthalene, alkylated naphthalenes or theirderivatives, methanol, ethanol, propanol, butanol, cyclohexanol,cyclohexanone, isophorone, highly polar solvents, for example dimethylsulfoxide, N-methylpyrrolidone or water.

Also anti-freezing agents such as glycerin, ethylene glycol, propyleneglycol and bactericides such as can be added to the formulation.

Suitable antifoaming agents are for example antifoaming agents based onsilicon or magnesium stearate.

Suitable preservatives are for example Dichlorophen andenzylalkoholhemiformal.

Seed Treatment formulations may additionally comprise binders andoptionally colorants.

Binders can be added to improve the adhesion of the active materials onthe seeds after treatment. Suitable binders are block copolymers EO/POsurfactants but also polyvinylalcoholsl, polyvinylpyrrolidones,polyacrylates, polymethacrylates, polybutenes, polyisobutylenes,polystyrene, polyethyleneamines, polyethyleneamides, polyethyleneimines(Lupasol®, Polymin®), polyethers, polyurethans, polyvinylacetate, tyloseand copolymers derived from these polymers.

Optionally, also colorants can be included in the formulation. Suitablecolorants or dyes for seed treatment formulations are Rhodamin B, C.I.Pigment Red 112, C.I. Solvent Red 1, pigment blue 15:4, pigment blue15:3, pigment blue 15:2, pigment blue 15:1, pigment blue 80, pigmentyellow 1, pigment yellow 13, pigment red 112, pigment red 48:2, pigmentred 48:1, pigment red 57:1, pigment red 53:1, pigment orange 43, pigmentorange 34, pigment orange 5, pigment green 36, pigment green 7, pigmentwhite 6, pigment brown 25, basic violet 10, basic violet 49, acid red51, acid red 52, acid red 14, acid blue 9, acid yellow 23, basic red 10,basic red 108.

An example of a suitable gelling agent is carrageen (Satiagel®).

Powders, materials for spreading, and dustable products can be preparedby mixing or concomitantly grinding the active substances with a solidcarrier.

Granules, for example coated granules, impregnated granules andhomogeneous granules, can be prepared by binding the active compounds tosolid carriers. Examples of solid carriers are mineral earths such assilica gels, silicates, talc, kaolin, attaclay, limestone, lime, chalk,bole, loess, clay, dolomite, diatomaceous earth, calcium sulfate,magnesium sulfate, magnesium oxide, ground synthetic materials,fertilizers, such as, for example, ammonium sulfate, ammonium phosphate,ammonium nitrate, ureas, and products of vegetable origin, such ascereal meal, tree bark meal, wood meal and nutshell meal, cellulosepowders and other solid carriers.

In general, the formulations comprise from 0.01 to 95% by weight,preferably from 0.1 to 90% by weight, of the AHAS-inhibiting herbicide.In this case, the AHAS-inhibiting herbicides are employed in a purity offrom 90% to 100% by weight, preferably 95% to 100% by weight (accordingto NMR spectrum). For seed treatment purposes, respective formulationscan be diluted 2-10 fold leading to concentrations in the ready to usepreparations of 0.01 to 60% by weight active compound by weight,preferably 0.1 to 40% by weight.

The AHAS-inhibiting herbicide can be used as such, in the form of theirformulations or the use forms prepared therefrom, for example in theform of directly sprayable solutions, powders, suspensions ordispersions, emulsions, oil dispersions, pastes, dustable products,materials for spreading, or granules, by means of spraying, atomizing,dusting, spreading or pouring. The use forms depend entirely on theintended purposes; they are intended to ensure in each case the finestpossible distribution of the AHAS-inhibiting herbicide according to theinvention.

Aqueous use forms can be prepared from emulsion concentrates, pastes orwettable powders (sprayable powders, oil dispersions) by adding water.To prepare emulsions, pastes or oil dispersions, the substances, as suchor dissolved in an oil or solvent, can be homogenized in water by meansof a wetter, tackifier, dispersant or emulsifier. However, it is alsopossible to prepare concentrates composed of active substance, wetter,tackifier, dispersant or emulsifier and, if appropriate, solvent or oil,and such concentrates are suitable for dilution with water.

The active compound concentrations in the ready-to-use preparations canbe varied within relatively wide ranges. In general, they are from0.0001 to 10%, preferably from 0.01 to 1% per weight.

The AHAS-inhibiting herbicide may also be used successfully in theultra-low-volume process (ULV), it being possible to apply formulationscomprising over 95% by weight of active compound, or even to apply theactive compound without additives.

The following are examples of formulations:

1. Products for dilution with water for foliar applications. For seedtreatment purposes, such products may be applied to the seed diluted orundiluted.

A) Water-soluble concentrates (SL, LS)

Ten parts by weight of the AHAS-inhibiting herbicide are dissolved in 90parts by weight of water or a water-soluble solvent. As an alternative,wetters or other auxiliaries are added. The AHAS-inhibiting herbicidedissolves upon dilution with water, whereby a formulation with 10% (w/w)of AHAS-inhibiting herbicide is obtained.

B) Dispersible concentrates (DC)

Twenty parts by weight of the AHAS-inhibiting herbicide are dissolved in70 parts by weight of cyclohexanone with addition of 10 parts by weightof a dispersant, for example polyvinylpyrrolidone. Dilution with watergives a dispersion, whereby a formulation with 20% (w/w) ofAHAS-inhibiting herbicide is obtained.

C) Emulsifiable concentrates (EC)

Fifteen parts by weight of the AHAS-inhibiting herbicide are dissolvedin 7 parts by weight of xylene with addition of calciumdodecylbenzenesulfonate and castor oil ethoxylate (in each case 5 partsby weight). Dilution with water gives an emulsion, whereby a formulationwith 15% (w/w) of AHAS-inhibiting herbicide is obtained.

D) Emulsions (EW, EO, ES)

Twenty-five parts by weight of the AHAS-inhibiting herbicide aredissolved in 35 parts by weight of xylene with addition of calciumdodecylbenzenesulfonate and castor oil ethoxylate (in each case 5 partsby weight). This mixture is introduced into 30 parts by weight of waterby means of an emulsifier machine (e.g. Ultraturrax) and made into ahomogeneous emulsion. Dilution with water gives an emulsion, whereby aformulation with 25% (w/w) of AHAS-inhibiting herbicide is obtained.

E) Suspensions (SC, OD, FS)

In an agitated ball mill, 20 parts by weight of the AHAS-inhibitingherbicide are comminuted with addition of 10 parts by weight ofdispersants, wetters and 70 parts by weight of water or of an organicsolvent to give a fine AHAS-inhibiting herbicide suspension. Dilutionwith water gives a stable suspension of the AHAS-inhibiting herbicide,whereby a formulation with 20% (w/w) of AHAS-inhibiting herbicide isobtained.

F) Water-dispersible granules and water-soluble granules (WG, SG)

Fifty parts by weight of the AHAS-inhibiting herbicide are ground finelywith addition of 50 parts by weight of dispersants and wetters and madeas water-dispersible or water-soluble granules by means of technicalappliances (for example extrusion, spray tower, fluidized bed). Dilutionwith water gives a stable dispersion or solution of the AHAS-inhibitingherbicide, whereby a formulation with 50% (w/w) of AHAS-inhibitingherbicide is obtained.

G) Water-dispersible powders and water-soluble powders (WP, SP, SS, WS)

Seventy-five parts by weight of the AHAS-inhibiting herbicide are groundin a rotor-stator mill with addition of 25 parts by weight ofdispersants, wetters and silica gel. Dilution with water gives a stabledispersion or solution of the AHAS-inhibiting herbicide, whereby aformulation with 75% (w/w) of AHAS-inhibiting herbicide is obtained.

I) Gel-Formulation (GF)

In an agitated ball mill, 20 parts by weight of the AHAS-inhibitingherbicide are comminuted with addition of 10 parts by weight ofdispersants, 1 part by weight of a gelling agent wetters and 70 parts byweight of water or of an organic solvent to give a fine AHAS-inhibitingherbicide suspension. Dilution with water gives a stable suspension ofthe AHAS-inhibiting herbicide, whereby a formulation with 20% (w/w) ofAHAS-inhibiting herbicide is obtained. This gel formulation is suitablefor us as a seed treatment.

2. Products to be applied undiluted for foliar applications. For seedtreatment purposes, such products may be applied to the seed diluted.

A) Dustable powders (DP, DS)

Five parts by weight of the AHAS-inhibiting herbicide are ground finelyand mixed intimately with 95 parts by weight of finely divided kaolin.This gives a dustable product having 5% (w/w) of AHAS-inhibitingherbicide.

B) Granules (GR, FG, GG, MG)

One-half part by weight of the AHAS-inhibiting herbicide is groundfinely and associated with 95.5 parts by weight of carriers, whereby aformulation with 0.5% (w/w) of AHAS-inhibiting herbicide is obtained.Current methods are extrusion, spray-drying or the fluidized bed. Thisgives granules to be applied undiluted for foliar use.

Conventional seed treatment formulations include for example flowableconcentrates FS, solutions LS, powders for dry treatment DS, waterdispersible powders for slurry treatment WS, water-soluble powders SSand emulsion ES and EC and gel formulation GF. These formulations can beapplied to the seed diluted or undiluted. Application to the seeds iscarried out before sowing, either directly on the seeds.

In a preferred embodiment a FS formulation is used for seed treatment.Typically, a FS formulation may comprise 1-800 g/l of active ingredient,1-200 g/l Surfactant, 0 to 200 g/l antifreezing agent, 0 to 400 g/l ofbinder, 0 to 200 g/l of a pigment and up to 1 liter of a solvent,preferably water.

The present invention non-transgenic and transgenic seeds of theherbicide-resistant plants of the present invention. Such seeds include,for example, non-transgenic rice seeds comprising theherbicide-tolerance characteristics of the plant with NCIMB AccessionNumber NCIMB 41262, and transgenic seeds comprising a polynucleotidemolecule of the invention that encodes an IMI protein.

For seed treatment, seeds of the herbicide resistant plants according ofthe present invention are treated with herbicides, preferably herbicidesselected from the group consisting of AHAS-inhibiting herbicides such asamidosulfuron, azimsulfuron, bensulfuron, chlorimuron, chlorsulfuron,cinosulfuron, cyclosulfamuron, ethametsulfuron, ethoxysulfuron,flazasulfuron, flupyrsulfuron, foramsulfuron, halosulfuron,imazosulfuron, iodosulfaron, mesosulfuron, metsulfuron, nicosulfuron,oxasulfuron, primisulfuron, prosulfuron, pyrazosulfuron, rimsulfuron,sulfometuron, sulfosulfuron, thifensulfuron, triasulfuron, tribenuron,trifloxysulfuron, triflusulfuron, tritosulfuron, imazamethabenz,imazamox, imazapic, imazapyr, imazaquin, imazethapyr, cloransulam,diclosulam, florasulam, flumetsulam, metosulam, penoxsulam, bispyribac,pyriminobac, propoxycarbazone, flucarbazone, pyribenzoxim, pyriftalid,pyrithiobac, and mixtures thereof, or with a formulation comprising aAHAS-inhibiting herbicide.

The term seed treatment comprises all suitable seed treatment techniquesknown in the art, such as seed dressing, seed coating, seed dusting,seed soaking, and seed pelleting.

In accordance with one variant of the present invention, a furthersubject of the invention is a method of treating soil by theapplication, in particular into the seed drill: either of a granularformulation containing the AHAS-inhibiting herbicide as acomposition/formulation (e.g. a granular formulation, with optionallyone or more solid or liquid, agriculturally acceptable carriers and/oroptionally with one or more agriculturally acceptable surfactants. Thismethod is advantageously employed, for example, in seedbeds of cereals,maize, cotton, and sunflower.

The present invention also comprises seeds coated with or containingwith a seed treatment formulation comprising at least oneAHAS-inhibiting herbicide selected from the group consisting ofamidosulfuron, azimsulfuron, bensulfuron, chlorimuron, chlorsulfuron,cinosulfuron, cyclosulfamuron, ethametsulfuron, ethoxysulfuron,flazasulfuron, flupyrsulfuron, foramsulfuron, halosulfuron,imazosulfuron, iodosulfuron, mesosulfuron, metsulfuron, nicosulfuron,oxasulfuron, primisulfuron, prosulfuron, pyrazosulfuron, rimsulfuron,sulfometuron, sulfosulfuron, thifensulfuron, triasulfuron, tribenuron,trifloxysulfuron, triflusulfuron, tritosulfuron, imazamethabenz,imazamox, imazapic, imazapyr, imazaquin, imazethapyr, cloransulam,diclosulam, florasulam, flumetsulam, metosulam, penoxsulam, bispyribac,pyriminobac, propoxycarbazone, flucarbazone, pyribenzoxim, pyriftalidand pyrithiobac.

The term seed embraces seeds and plant propagules of all kinds includingbut not limited to true seeds, seed pieces, suckers, corms, bulbs,fruit, tubers, grains, cuttings, cut shoots and the like and means in apreferred embodiment true seeds.

The term “coated with and/or containing” generally signifies that theactive ingredient is for the most part on the surface of the propagationproduct at the time of application, although a greater or lesser part ofthe ingredient may penetrate into the propagation product, depending onthe method of application. When the said propagation product is(re)planted, it may absorb the active ingredient.

The seed treatment application with the AHAS-inhibiting herbicide orwith a formulation comprising the AHAS-inhibiting herbicide is carriedout by spraying or dusting the seeds before sowing of the plants andbefore emergence of the plants.

In the treatment of seeds, the corresponding formulations are applied bytreating the seeds with an effective amount of the AHAS-inhibitingherbicide or a formulation comprising the AHAS-inhibiting herbicide.Herein, the application rates are generally from 0.1 g to 10 kg of thea.i. (or of the mixture of a.i. or of the formulation) per 100 kg ofseed, preferably from 1 g to 5 kg per 100 kg of seed, in particular from1 g to 2.5 kg per 100 kg of seed. For specific crops such as lettuce therate can be higher.

The present invention provides a method for combating undesiredvegetation or controlling weeds comprising contacting the seeds of theresistant plants according to the present invention before sowing and/orafter pregermination with an AHAS-inhibiting herbicide. The method canfurther comprise sowing the seeds, for example, in soil in a field or ina potting medium in greenhouse. The method finds particular use incombating undesired vegetation or controlling weeds in the immediatevicinity of the seed.

The control of undesired vegetation is understood as meaning the killingof weeds and/or otherwise retarding or inhibiting the normal growth ofthe weeds. Weeds, in the broadest sense, are understood as meaning allthose plants which grow in locations where they are undesired.

The weeds of the present invention include, for example, dicotyledonousand monocotyledonous weeds. Dicotyledonous weeds include, but are notlimited to, weeds of the genera: Sinapis, Lepidium, Galium, Stellaria,Matricaria, Anthemis, Galinsoga, Chenopodium, Urtica, Senecio,Amaranthus, Portulaca, Xanthium, Convolvulus, Ipomoea, Polygonum,Sesbania, Ambrosia, Cirsium, Carduus, Sonchus, Solanum, Rorippa, Rotala,Lindernia, Lamium, Veronica, Abutilon, Emex, Datura, Viola, Galeopsis,Papaver, Centaurea, Trifolium, Ranunculus, and Taraxacum.Monocotyledonous weeds include, but are not limited to, weeds of of thegenera: Echinochloa, Setaria, Panicum, Digitaria, Phleum, Poa, Festuca,Eleusine, Brachiaria, Lolium, Bromus, Avena, Cyperus, Sorghum,Agropyron, Cynodon, Monochoria, Fimbristyslis, Sagittaria, Eleocharis,Scirpus, Paspalum, Ischaemum, Sphenoclea, Dactyloctenium, Agrostis,Alopecurus, and Apera.

In addition, the weeds of the present invention can include, forexample, crop plants that are growing in an undesired location. Forexample, a volunteer maize plant that is in a field that predominantlycomprises soybean plants can be considered a weed, if the maize plant isundesired in the field of soybean plants.

The articles “a” and “an” are used herein to refer to one or more thanone (i.e., to at least one) of the grammatical object of the article. Byway of example, “an element” means one or more elements.

As used herein, the word “comprising,” or variations such as “comprises”or “comprising,” will be understood to imply the inclusion of a statedelement, integer or step, or group of elements, integers or steps, butnot the exclusion of any other element, integer or step, or group ofelements, integers or steps.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLE 1 Production of an Imidazolinone-Resistant Rice Line

AHASL is a nuclear encoded enzyme and its gene, in different species,has been sequenced in its wild form and other forms showing sites ofmutation that confer resistance to sulfonylurea and imidazolinoneherbicides. In order to produce rice plants with herbicide-resistantAHAS enzymes, rice seeds were treated with a chemical mutagen in anattempt to induce small changes at the active site of interaction withthe herbicide in order to prevent inhibition. Leading rice varieties andelite rice lines were selected in order to generate a new mutation thatis resistant to the most active imidazolinones in excellent germplasm.Selection pressure was based on exposure of four-leaf stage plants totwo of the most active imidazolinone herbicides applied in oneapplication during several generations until homozygous highly resistantlines were obtained. The imidazolinone-resistance rice lines wereproduced as described below.

In the late spring of growing season number 1, two samples of seeds (600g each) of the rice cultivar IRGA 417 were treated with a 0.001 M sodiumazide aqueous solution at pH 3 (phosphate buffer 0.067M). This treatmentwas applied by soaking each seed-sample in a two-liter Erlenmeyercontaining one liter of the sodium azide solution, under constantshaking, for 18 hours, at room temperature. After treatment, the seedswere rinsed in tap water and, later on, they were partiallydried-aerated on blotting paper sheets in order to extract the moisturefrom the seeds surface. Afterwards, treated seeds were directly sown atthe field nursery in Concepcion del Uruguay, E.R., Argentina.

Treated (M₁) and untreated control seeds of the rice cultivar IRGA 417were planting in the field nursery a rate of 50 plants per square meter.The plants were grown under flooded conditions until maturity (26% grainmoisture) and bulk harvested. Seeds (M₂) of the plants were collectedand dried in a convector drier for 14 h at 45° C. They were kept inclose storage until next growing season.

In the late spring of growing season number 2, M₂ seeds were plantedwith an experimental seed planter for large areas at a rate of 50 kg/hain at the field nursery in Concepcion del Uruguay, E.R., Argentina. Anarea of 3 ha was established comprising a population of approximately6×10⁶ M₂ plants. IRGA 417 (wild type) was also planted as a control. Theentire area was subjected to a selection pressure with a mixture of twoimidazolinone herbicides. Three separate applications were done with acommercial sprayer in different directions to prevent any escape andresulting in a 3× treatment. A total volume of 222 L/ha was sprayed at50 psi, with Teejets 8002 nozzles, in each application. The rate of the1× treatment was a mixture of Arsenal® (Imazapir 75 cc a.i/ha) andCadre® (Imazapic 24.85 cc a.i/ha) in a water solution with a non-ionicsurfactant (Citowett) at the rate of 0.25%. The applications were doneat the four leaf-stage of the rice plants. No rainfall was registeredduring the 7 days after treatments.

Observations at regular times were done to survey the entireherbicide-treated area. At 90 days after the herbicide treatment, thesurviving individuals were labeled and transplanted to the greenhousefor asexual multiplication and seed production. A total of 10 individualplants were grown, and the seed was harvested and dried in a seedincubator for 7 days at 50° C. As expected, none of the control plants(IRGA 417) survived the herbicide treatment.

Seeds (M₃) from selected M₂ plants were planted in individual pots undergreenhouse conditions in Concepcion del Uruguay, E.R., Argentina duringthe winter immediately following the second season. A 2× treatment wasapplied with a backpack sprayer divided in two applications of a 1× rateof Arsenal® (Imazapir 75 cc a.i/ha) and Cadre® (Imazapic 24.85 cca.i/ha) in a water solution with a non-ionic surfactant (Citowet) at therate of 0.25%.

Tillers from the herbicide-tolerant plants (e.g., plants that survivedherbicide treatments) were grown until maturity (26% grain moisture) andhand harvested. The harvested seed (M₄) was subjected to a dormancybreaking treatment of 7 days at 50° C. and prepared for the next growingseason planting. Seeds from two herbicide-tolerant plants were keptseparately. The seeds from were prepared for a late-season plantingoutdoors at Concepción del Uruguay, E.R., Argentina.

In the summer of growing season number 3, M₄ seeds of anherbicide-tolerant M₃ plant that was designated as IMINTA 16 wereplanted with at the rate of 50 kg/ha. A treatment of 2× of imidazolinoneherbicides was applied at the four to five leaves stage of the riceplants as two applications of a 1× rate of Arsenal® (Imazapir 75 cca.i/ha) and Cadre® (Imazapic 24.85 cc a.i/ha) in a water solution with anon-ionic surfactant (Citowet) at the rate of 0.25%. No phytotoxicsymptoms were observed for plants of the IMINTA 16 line. No wild-typesegregants (i.e., not tolerant to the herbicide treatment) wereobserved, and a highly homogenous population in agronomic and tolerancetraits had been produced. Individual IMINTA 16 plants were transplantedinto the greenhouse for seed production, and seeds (M₅) were harvestedlater that year.

EXAMPLE 2 An Imidazoline-Resistant Rice Line with a Mutation in theAHASL1 Gene

Genomic DNA was separately extracted from leaves of greenhouse-grownseedlings of the IMINTA 16 line described in Example 1 above and theAHASL1 gene was amplified by a polymerase chain reaction (PCR) methodusing the primers described below. The resulting products of theindividual PCR amplifications were sequenced using standard methods.

The primers used for PCR and sequencing are provided in Table 1. Theprimers were selected manually by visual inspection of the publiclyknown rice sequences for Oryza sativa ‘Kinmaze’, Oryza sativa japonicaand Oryza sativa indica. The primers were nested approximately every400-500 bp along the approximately 2000 bp of the AHAS gene. Severalprimers were designed for each 500 bp stretch to maximize the likelihoodof success of amplification. No weight was given to conserved regionswhen choosing primers. The primer sequences were checked for hairpinsand dimers via the website wwvv.rnature.com/oligonucleotide.html.Because there are no introns present in the AHASL1 gene, the entire generepresents coding sequence, and is therefore conserved. Primers weredesigned to have a GC content close to 50% and similar meltingtemperatures, approximately 54-58° C. The primer names in Table 1reflect the exact starting base position according to public AHASL1nucleotide sequences for rice (e.g., GenBank Accession No. AB049822).U136851 refers to a region upstream of the start codon in the AHAS geneand was designed from a BAC clone (OSJNBa0053B21), accession no.AL731599.

TABLE 1 Primers for PCR Amplification and DNA Sequencingof the Rice AHASL1 Gene in IMINTA 16 Melting GC Primer Temp. contentName Sequence 5′ to 3′ (° C.) (%) Pair 1 U136851 GACATATGGGGCCCACTGT58.8 58   (SEQ ID NO: 4) L789 GTAGATTCATCGAGGTGTC 54.5 47.4(SEQ ID NO: 5) Pair 2 U642* GTCCTTGATGTGGAGGACAT 57.3 50  (SEQ ID NO: 6) L1369 CATATTGCGGTGGGATCTCT 57.3 50   (SEQ ID NO: 7)Pair 3 U1229 GGGCTTGAATGCTCTGCTAC 59.4 55   (SEQ ID NO: 8) L1742CGGGTTGCCCAAGTATGTAT 57.3 50   (SEQ ID NO: 9) Pair 4 U1633ACCTCCCTGTGAAGGTGATG 59.4 55   (SEQ ID NO: 10) L2155*AGGATTACCATGCCAAGCAC 57.3 50   (SEQ ID NO: 11)Alternate PCR primers and sequencing primers U037 CACCACCCACCATGGCTA58.2 61.1 (SEQ ID NO: 12) U114 GTAAGAACCACCAGCGAC 56   55.6(SEQ ID NO: 13) U1109 GTGGATAAGGCTGACCTGT 56.7 52.6 (SEQ ID NO: 14)U1166 GGGAAAATTGAGGCTTTTGCA 55.9 42.9 (SEQ ID NO: 15) L1299CTCATTGTGCCATGCACTAA 55.3 45   (SEQ ID NO: 16) U1721 GCATACATACTTGGGCAAC54   47   (SEQ ID NO: 17) L2054 CATACCACTCTTTATGGGTC 52   45  (SEQ ID NO: 18) *Also used U642 and L2155 as a PCR pair

When the PCR-amplified genomic DNA from the IMINTA 16 seedlings wasexamined, a single base change (i.e., transition, C to T) was identifiedin the coding region of the gene that caused an amino acid substitutionin the AHASL1 protein at amino acid position 179 from Ala in the wildtype line to Val in the IMINTA 16 line. The site of this substitutioncorresponds to position 205 in the Arabidopsis thaliana AHASL protein(see, Table 2 below). The Ala205Val substitution in the Arabidopsisthaliana AHASL protein is known to confer on plants that express thisprotein tolerance to imidazolinone herbicides.

EXAMPLE 3 Herbicide-Resistant Rice AHASL1 Proteins

The present invention discloses both the nucleotide and amino acidsequences for herbicide resistant rice AHASL1 polypeptides. Plantscomprising herbicide-resistant AHASL1 polypeptides have been previouslyidentified, and a number of conserved regions of AHASL1 polypeptidesthat are the sites of amino acids substitutions that confer herbicideresistance have been described. See, Devine and Eberlein (1997)“Physiological, biochemical and molecular aspects of herbicideresistance based on altered target sites”. In: Herbicide Activity:Toxicology, Biochemistry and Molecular Biology, Roe et al. (eds.), pp.159-185, IOS Press, Amsterdam; and Devine and Shukla, (2000) CropProtection 19:881-889.

Using the AHASL1 polynucleotide molecules of the invention and methodsknown to those of ordinary skill in art, one can produce additionalpolynucleotide molecules encoding herbicide resistant AHASL1polypeptides having one, two, three, or more amino acid substitutions atthe identified sites in these conserved regions. Table 2 provides theconserved regions of AHASL1 proteins, the amino acid substitutions knownto confer herbicide resistance within these conserved regions, and thecorresponding amino acids in the rice AHASL1 protein set forth in SEQ IDNO: 2.

TABLE 2 Amino Acid Substitutions in Conserved Regions of AHASLPolypeptides that are Known to Confer Herbicide-Resistance and theirEquivalent Position in Rice AHASL1 Polypeptides Amino acid positionConserved region¹ Mutation² Reference in rice VFAYPGG A SMEIHQALTRS³Ala₁₂₂ to Thr Bernasconi et al.⁴ Ala₉₆ AITGQV P RRMIGT³ Pro₁₉₇ to AlaBoutsalis et al.⁵ Pro₁₇₁ ¹² Pro₁₉₇ to Thr Guttieri et al.⁶ Pro₁₉₇ to HisGuttieri et al.⁷ Pro₁₉₇ to Leu Guttieri et al.⁶ Pro₁₉₇ to Arg Guttieriet al.⁶ Pro₁₉₇ to Ile Boutsalis et al.⁶ Pro₁₉₇ to Gln Guttieri et al.⁶Pro₁₉₇ to Ser Guttieri et al.⁶ A FQETP³ Ala₂₀₅ to Asp Hartnett et al.⁸Ala₁₇₉ ¹³ Ala₂₀₅ to Val¹¹ Simpson⁹ Q W ED³ Trp₅₇₄ to Leu Bruniard¹⁰Trp₅₄₈ Boutsalis et al.⁵ IP S GG³ Ser₆₅₃ to Asn Chang & Ser₆₂₇Duggleby¹² Ser₆₅₃ to Thr Lee et al.¹³ Ser₆₅₃ to Phe ¹Conserved regionsfrom Devine and Eberlein (1997) “Physiological, biochemical andmolecular aspects of herbicide resistance based on altered targetsites”. In: Herbicide Activity: Toxicology, Biochemistry and MolecularBiology, Roe et al. (eds.), pp. 159-185, IOS Press, Amsterdam and Devineand Shukla, (2000) Crop Protection 19: 881-889. ²Amino acid numberingcorresponds to the amino acid sequence of the Arabidopsis thaliana AHASLpolypeptide. ³The rice AHASL1 protein of the invention (SEQ ID NO: 2)has the same conserved regions. ⁴Bernasconi et al. (1995) J. Biol Chem.270(29): 17381-17385. ⁵Boutsalis et al. (1999) Pestic. Sci. 55: 507-516.⁶Guttieri et al. (1995) Weed Sci. 43: 143-178. ⁷Guttieri et al. (1992)Weed Sci. 40: 670-678. ⁸Hartnett et al. (1990) “Herbicide-resistantplants carrying mutated acetolactate synthase genes,” In: ManagingResistance to Agrochemicals: Fundamental Research to PracticalStrategies, Green et al. (eds.), American Chemical Soc. Symp., SeriesNo. 421, Washington, DC, USA ⁹Simpson (1998) Down to Earth 53(1): 26-35.¹⁰Bruniard (2001) Inheritance of imidazolinone resistance,characterization of cross-resistance pattern, and identification ofmolecular markers in sunflower (Helianthus annuus L.). Ph.D. Thesis,North Dakota State University, Fargo, ND, USA, pp 1-78. ¹¹The presentinvention discloses the amino acid sequence of a herbicide-resistantrice AHASL1 protein with the Ala₁₇₉ to Val substitution (SEQ ID NO: 2)and a polynucleotide sequences encoding this herbicide resistant AHASL1(SEQ ID NOS: 1 and 3). ¹²Chang and Duggleby (1998) Biochem J. 333:765-777. ¹³Lee et al. (1999) FEBS Lett. 452: 341-345.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. A method for treating rice, comprising: providinga rice crop plant and at least one AHAS-inhibiting herbicide; applyingan effective amount of the at least one AHAS-inhibiting herbicide to therice crop plant, post-emergence; thereby creating a treated rice plant;and growing the resulting treated rice plant.
 2. The method of claim 1,further comprising harvesting seed from the treated rice plant.
 3. Themethod of claim 1, wherein the rice crop plant comprises in its genomeat least one copy of a rice acetohydroxyacid synthase large subunit(AHASL1) polynucleotide that encodes a herbicide-tolerant AHASL1protein, and wherein said herbicide-tolerant AHASL1 protein provides therice crop plant with increased tolerance to at least one AHAS-inhibitingherbicide as compared to a wild-type rice crop plant.
 4. The method ofclaim 3, wherein the AHASL1 protein comprises a leucine substitution ata position corresponding to position 171 of SEQ ID NO:2.
 5. The methodof claim 1, wherein the AHAS-inhibiting herbicide comprises one or moreof an imidazolinone herbicide, a sulfonylurea herbicide, atriazolopyrimidine herbicide, a pyrimidinyloxybenzoate herbicide, and asulfonylaminocarbonyltriazolinone herbicide.
 6. The method of claim 5,wherein the AHAS-inhibiting herbicide comprises a sulfonylureaherbicide.
 7. The method of claim 6, wherein the sulfonylurea herbicidecomprises one or more of chlorsulfuron, metsulfuron, metsulfuron methyl,sulfometuron, sulfometuron methyl, chlorimuron, chlorimuron ethyl,thifensulfuron, thifensulfuron methyl, tribenuron, tribenuron methyl,bensulfuron, bensulfuron methyl, nicosulfuron, ethametsulfuron,ethametsulfuron methyl, rimsulfuron, triflusulfuron, triflusulfuronmethyl, triasulfuron, primisulfuron, primisulfuron methyl, cinosulfuron,amidosulfiuon, fluzasulfuron, imazosulfuron, pyrazosulfuron,pyrazosulfuron ethyl, halosulfuron, azimsulfuron, cyclosulfuron,ethoxysulfuron, flazasulfuron, flupyrsulfuron, flupyrsulfuron methyl,foramsulfuron, iodosulfuron, oxasulfuron, mesosulfuron, prosulfuron,sulfosulfuron, trifloxysulfuron, tritosulfuron, and derivatives thereof.8. The method of claim 7, wherein the sulfonylurea herbicide comprisesone or more of chlorsulfuron, flazasulfuron, flucetosulfuron,flupyrsulfuron, flupyrsulfuron methyl, foramsulfuron, mesosulfuron,nicosulfuron, primisulfuron, primisulfuron methyl, rimsulfuron,sulfometuron, sulfometuron methyl, sulfusulfuron, and derivativesthereof.
 9. The method of claim 5, wherein the AHAS-inhibiting herbicidecomprises an imidazolinone herbicide.
 10. The method of claim 9, whereinthe imidazolinone herbicide comprises one or more of imazethapyr,imazapic, imazamox, imazaquin, imazethabenz, imazapyr, and derivativesthereof.
 11. The method of claim 9, wherein the imidazolinone herbicidecomprises imazethapyr.
 12. The method of claim 1, wherein the effectiveamount is effective for killing a weed of one or more of the generaEchinochloa, Setaria, Panicum, Digitaria, Phleum, Poa, Festuca,Eleusine, Brachiaria, Lolium, Bromus, Avena, Cyperus, Sorghum,Agropyron, Cynodon, Monochoria, Fimbristyslis, Sagittaria, Eleocharis,Scirpus, Paspalum, Ischaemum, Sphenoclea, Dactyloctenium, Agrostis,Alopecurus, and Apera.
 13. The method of claim 12, wherein the effectiveamount is effective for killing a weed of one or more of the generaDigitaria, Eleusine, Ischaemum, Paspalum, and Eleocharis.
 14. The methodof claim 1, wherein the effective amount is effective for killing redrice.